Variant thioesterases and methods of use

ABSTRACT

The present invention relates to variant thioesterases and their use in plants, e.g., to increase enzymatic activity and to promote increased production of mid-chain length fatty acids (e.g., 8 to 14 carbons) and at desired ratios. Further disclosed herein are methods of manufacturing renewable chemicals through the manufacture of novel triglyceride oils followed by chemical modification of the oils. Oils containing fatty acid chain lengths of C8, C10, C12 or C14 are also disclosed and are useful as feedstocks in the methods described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 62/028,641, filed on Jul. 24, 2014, which ishereby incorporated herein by reference in its entirety for allpurposes. This application is technologically related to the subjectmatter of PCT/US2014/013676, entitled “Variant Thioesterases and Methodsof Use,” and filed Jan. 29, 2014, which is hereby incorporated herein byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 5, 2015, isnamed SOLAP027US_SL.txt and is 180,154 bytes in size.

FIELD

The present invention relates to variant acyl-ACP thioesterases andtheir use in oil-producing cells, e.g., to increase enzymatic activitytoward certain acyl-ACP substrates and to promote increased productionof oils with desired fatty acid profiles.

BACKGROUND

Today, fats and fatty acids primarily come from vegetable and animalsources, with the notable exception of commercial production of omega-3fatty acids by fermentation of microbes for use in baby formula andnutritional supplements. Progress is being made however toward thecommercial production of tailored oils using recombinant microalgae. SeePCT Publications WO2008/151149, WO2010/06032, WO2011/150410,WO2011/150411, and international patent application PCT/US12/23696.

One method for producing a desired fatty acid profile in an oleaginousorganism is to introduce an acyl-ACP thioesterase transgene; e.g., atransgene from a plant that produces a desired fatty acid.

By terminating fatty acid biosynthesis, the acyl-acyl carrier protein(ACP) thioesterase (TE) functionally determines the length and identityof the fatty acid end product (Salas et al., (2002) Archives ofBiochemistry and Biophysics 403: 25-34). Based on amino acid sequencealignments, the plant TEs have been shown to cluster into two families,FatAs, which show marked preference for 18:1-ACP with minor activitytowards 18:0- and 16:0-ACPs; and FatBs, which hydrolyze primarilysaturated acyl-ACPs with chain lengths that vary between 8-16 carbons(Voelker, In Genetic Engineering Volume 18. Edited by: Setlow JK. NewYork, Plenum Press; 1996:111-133; Ginalski, et al., Nucl Acids Res(2003) 31:3291-3292; and Jones, et al., (1995) Plant Cell 7: 359-371).FatB TEs have a conserved hydrophobic 18-amino acid domain (Facciottiand Yuan (1998) European Journal of Lipid Science and Technology100:167-172), and a conserved Asn-His-Cys catalytic triad in theC-terminal catalytic domain (Blatti, et al., PLoS ONE (2012) 7(9):e42949. doi:10.1371 and Mayer and Shanklin, BMC Plant Biology (2007)7:1-11). Mayer and Shanklin, BMC Plant Biology (2007) 7:1-11, identify aC-terminal conserved acyl-ACP thioesterase catalytic domaincharacterized by a C-terminal hot dog fold encompassing the Cys-His-Asncatalytic triad.

SUMMARY

Provided is a non-natural protein, an isolated gene encoding thenon-natural protein, an expression cassette expressing the non-naturalprotein, or a host cell comprising the expression cassette. In someembodiments, the non-natural protein has at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 andcomprises Tyrosine (Y) or Phenylalanine (F) at the positioncorresponding to position 163 of SEQ ID NO: 1 and/or Proline (P), Lysine(K), or Alanine (A) at the position corresponding to position 186 of SEQID NO: 1. In some embodiments, the non-natural protein further comprisesa Lysine (K) at the position corresponding to position 228 of SEQ ID NO:1.

In a related aspect, provided is a method for producing a triglycerideoil. In varying embodiments, the methods comprise expressing, in a hostcell, the protein of mentioned immediately above, or a proteincomprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identity one of SEQ ID NOs: 3-8 that has Y or F at theposition corresponding to position 163 of SEQ ID NO: 1 and/or P, K, or Aat the position corresponding to position 186 of SEQ ID NO: 1. In someembodiments, the non-natural protein further comprises K at the positioncorresponding to position 228 of SEQ ID NO: 1. The method furtherincludes cultivating the host cell and isolating the oil.

In another aspect, provided is a method for increasing the C8 and/or C10fatty acids in a fatty acid profile of an oil produced by an optionallyoleaginous host cell. The method includes, providing a parent geneencoding a FATB enzyme, mutating the gene to so as to have Y or F at theposition corresponding to position 163 of SEQ ID NO: 1 and/or P, K, or Aat the position corresponding to position 186 of SEQ ID NO: 1. In someembodiments, the non-natural protein further comprises K at the positioncorresponding to position 228 of SEQ ID NO: 1. In varying embodiments,the method further includes expressing the mutated gene in the host celland producing the oil. The fatty acid profile of the oil is therebyincreased in C8 and/or C10 fatty acids relative to the parent gene.Optionally, the gene encoding the FATB enzyme encodes a protein with atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity to SEQ ID NO: 1, 13 or 14.

In an embodiment, provided is a non-natural protein, an isolated geneencoding the non-natural protein, an expression cassette expressing thenon-natural protein, or a host cell comprising the expression cassette.In varying embodiments, the non-natural protein has at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ IDNO: 13 and A or K at the position corresponding to position 230 of SEQID NO: 13. A method for producing an oil includes expressing, in a hostcell, the non-natural proteins described herein, cultivating the cell,and isolating the oil.

In another aspect, provided is a non-natural protein, an isolated geneencoding the non-natural protein, an expression cassette expressing thenon-natural protein, or a host cell comprising the expression cassette.In some embodiments, the non-natural protein has at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 45and comprises A, T or V at the position corresponding to position 74 ofSEQ ID NO: 45 (G96 of wild-type Gm FATA) and/or F, K or S at theposition corresponding to position 69 of SEQ ID NO: 45 (L91 of wild-typeGm FATA), and/or F, A, K or V at the position corresponding to position134 of SEQ ID NO: 45 (T156 of wild-type Gm FATA). In some embodiments,the non-natural protein further comprises A or V at the positioncorresponding to position 89 of SEQ ID NO: 45 (S111 of wild-type GmFATA) and/or A at the position corresponding to position 171 of SEQ IDNO: 45 (V193 of wild-type Gm FATA), and/or A or V at the positioncorresponding to position 86 of SEQ ID NO: 45 (G108 of wild-type GmFATA).

In a further aspect, provided is a method for producing a triglycerideoil. In various embodiments, the method comprises expressing, in a hostcell, the protein of claim 7 or claim 8, or a protein comprising atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity to one of SEQ ID NOs: 45 and 15-29 and comprises A, T or V atthe position corresponding to position 74 of SEQ ID NO: 45 (G96 ofwild-type Gm FATA) and/or F, K or S at the position corresponding toposition 69 of SEQ ID NO: 45 (L91 of wild-type Gm FATA), and/or F, A, Kor V at the position corresponding to position 134 of SEQ ID NO: 45(G156 of wild-type Gm FATA); cultivating the host cell; and isolatingthe oil. In some embodiments, the protein further comprises A or V atthe position corresponding to position 89 of SEQ ID NO: 45 (S111 ofwild-type Gm FATA) and/or A at the position corresponding to position171 of SEQ ID NO: 45 (V193 of wild-type Gm FATA), and/or A or V at theposition corresponding to position 86 of SEQ ID NO: 45 (G108 ofwild-type Gm FATA).

In another aspect, provided is a method for increasing the C18:0 fattyacids in a fatty acid profile of an oil produced by an optionallyoleaginous host cell. In some embodiments, the method further comprisesproviding a parent gene encoding a FATB enzyme, mutating the gene to soas to have A, T or V at the position corresponding to position 74 of SEQID NO: 45 (G96 of wild-type Gm FATA) and/or F, K or S at the positioncorresponding to position 69 of SEQ ID NO: 45 (L91 of wild-type GmFATA), and/or F, A, K or V at the position corresponding to position 134of SEQ ID NO: 45 (T156 of wild-type Gm FATA); expressing the mutatedgene in the host cell; and producing the oil, whereby the fatty acidprofile of the oil is increased in C18:0 fatty acids relative to theparent gene. In various embodiments, the method entails further mutatingthe gene to so as to have A or V at the position corresponding toposition 89 of SEQ ID NO: 45 (S111 of wild-type Gm FATA) and/or A at theposition corresponding to position 171 of SEQ ID NO: 45 (V193 ofwild-type Gm FATA), and/or A or V at the position corresponding toposition 86 of SEQ ID NO: 45 (G108 of wild-type Gm FATA). In someembodiments, the gene encoding the FATB enzyme encodes a protein with atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity to one of SEQ ID NOs: 45 and 15-29.

Definitions

An “acyl-ACP thioesterase” or “acyl-ACP TE” interchangeably refer to anenzyme that catalyzes the cleavage of a fatty acid from an acyl carrierprotein (ACP) during lipid synthesis. Acyl-acyl carrier protein (ACP)thioesterases (TEs) hydrolyze acyl-ACP thioester bonds, releasing freefatty acids and ACP.

The term “acyl-ACP preferring TE” refers to the fatty acyl-ACP substratespecificity of a TE. An acyl-ACP preferring TE preferentially liberatesa particular fatty acid from an acyl-ACP substrate. For example, theacyl-ACP preferring TE can preferentially liberate a given fatty acidover all other fatty acids in the set of C8:0, C10:0, C12:0, C14:0,C16:0, C18:0, C18:1, and C18:2 fatty acids. The preference of theacyl-ACP preferring TE can be detected as a higher V_(max) (or a higherk_(cat), or a higher V/K) in comparison to other non-preferred fattyacid-ACP substrates. The preference can be inferred from changes infatty acid profile of a cell genetically engineered to overexpress theacyl-ACP preferring TE relative to a control cell that does notoverexpress the acyl-ACP preferring TE.

Numbering of a given amino acid polymer or nucleic acid polymer“corresponds to” or is “relative to” the numbering of a selected aminoacid polymer or nucleic acid polymer when the position of any givenpolymer component (e.g., amino acid, nucleotide, also referred togenerically as a “residue”) is designated by reference to the same or toan equivalent position (e.g., based on an optimal alignment or aconsensus sequence) in the selected amino acid or nucleic acid polymer,rather than by the actual numerical position of the component in thegiven polymer.

A “variant” is a polypeptide comprising a sequence which differs in oneor more amino acid position(s) from that of a parent polypeptidesequence (e.g., by substitution, deletion, or insertion). A variant maycomprise a sequence which differs from the parent polypeptides sequencein up to 40% of the total number of residues of the parent polypeptidesequence, such as in up to 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%,7%, 6%, 5%, 4%, 3% 2% or 1% of the total number of residues of theparent polypeptide sequence. For example, a variant of a 400 amino acidpolypeptide sequence comprises a sequence which differs in up to 40% ofthe total number of residues of the parent polypeptide sequence, thatis, in up to 160 amino acid positions within the 400 amino acidpolypeptide sequence (such as in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 78, 79, 80, 85, 90, 95, 100, 105,110, 115, 120, 125, 130, 135, 140, 145, 150, 155, or 160 amino acidpositions within the reference sequence.

“Naturally occurring” as applied to a composition that can be found innature as distinct from being artificially produced by man. For example,a polypeptide or polynucleotide that is present in an organism(including viruses, bacteria, protozoa, insects, plants or mammaliantissue) that can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory is naturallyoccurring. “Non-naturally occurring” (also termed “synthetic” or“artificial”) as applied to an object means that the object is notnaturally-occurring—i.e., the object cannot be found in nature asdistinct from being artificially produced by man.

A “cell oil” or “cell fat” shall mean a predominantly triglyceride oilobtained from an organism, where the oil has not undergone blending withanother natural or synthetic oil, or fractionation so as tosubstantially alter the fatty acid profile of the triglyceride. Inconnection with an oil comprising triglycerides of a particularregiospecificity, the cell oil or cell fat has not been subjected tointeresterification or other synthetic process to obtain thatregiospecific triglyceride profile, rather the regiospecificity isproduced naturally, by a cell or population of cells. For a cell oil orcell fat produced by a cell, the sterol profile of oil is generallydetermined by the sterols produced by the cell, not by artificialreconstitution of the oil by adding sterols in order to mimic the celloil. In connection with a cell oil or cell fat, and as used generallythroughout the present disclosure, the terms oil and fat are usedinterchangeably, except where otherwise noted. Thus, an “oil” or a “fat”can be liquid, solid, or partially solid at room temperature, dependingon the makeup of the substance and other conditions. Here, the term“fractionation” means removing material from the oil in a way thatchanges its fatty acid profile relative to the profile produced by theorganism, however accomplished. The terms “cell oil” and “cell fat”encompass such oils obtained from an organism, where the oil hasundergone minimal processing, including refining, bleaching and/ordegumming, which does not substantially change its triglyceride profile.A cell oil can also be a “noninteresterified cell oil”, which means thatthe cell oil has not undergone a process in which fatty acids have beenredistributed in their acyl linkages to glycerol and remain essentiallyin the same configuration as when recovered from the organism.

A “fatty acid profile” is the distribution of fatty acyl groups in thetriglycerides of the oil without reference to attachment to a glycerolbackbone. Fatty acid profiles are typically determined by conversion toa fatty acid methyl ester (FAME), followed by gas chromatography (GC)analysis with flame ionization detection (FID). The fatty acid profilecan be expressed as one or more percent of a fatty acid in the totalfatty acid signal determined from the area under the curve for thatfatty acid. FAME-GC-FID measurement approximate weight percentages ofthe fatty acids.

“Microalgae” are microbial organisms that contain a chloroplast orplastid, and optionally that is capable of performing photosynthesis, ora prokaryotic microbial organism capable of performing photosynthesis.Microalgae include obligate photoautotrophs, which cannot metabolize afixed carbon source as energy, as well as heterotrophs, which can livesolely off of a fixed carbon source. Microalgae include unicellularorganisms that separate from sister cells shortly after cell division,such as Chlamydomonas, as well as microbes such as, for example, Volvox,which is a simple multicellular photosynthetic microbe of two distinctcell types. Microalgae include eukaryotic Chlorophyceae such asChlorella, Dunaliella, and Prototheca. Microalgae also include othermicrobial photosynthetic organisms that exhibit cell-cell adhesion, suchas Agmenellum, Anabaena, and Pyrobotrys. Microalgae also includeobligate heterotrophic microorganisms that have lost the ability toperform photosynthesis, such as certain dinoflagellate algae species andspecies of the genus Prototheca.

An “oleaginous” cell is a non-human cell capable of producing at least20% lipid by dry cell weight, naturally or through recombinant orclassical strain improvement. An “oleaginous microbe” or “oleaginousmicroorganism is a microbe, including a microalga that is oleaginous.

As used with respect to polypeptides or polynucleotides, the term“isolated” refers to a polypeptide or polynucleotide that has beenseparated from at least one other component that is typically presentwith the polypeptide or polynucleotide. Thus, a naturally occurringpolypeptide is isolated if it has been purified away from at least oneother component that occurs naturally with the polypeptide orpolynucleotide. A recombinant polypeptide or polynucleotide is isolatedif it has been purified away from at least one other component presentwhen the polypeptide or polynucleotide is produced.

The terms “polypeptide” and “protein” are used interchangeably herein torefer a polymer of amino acids, and unless otherwise limited, includeatypical amino acids that can function in a similar manner to naturallyoccurring amino acids.

The term “sequence”, as used in connection with a polypeptide or nucleicacid polymer refers to the order of monomers making up the polymer orthe sub-polymer or fragment having that sequence.

A “subsequence” of an amino acid or nucleotide sequence is a portion ofa larger sequence or the peptide or nucleic acid sub-polymer or fragmentcharacterized by the portion of the larger sequence.

The terms “identical” or “percent identity,” in the context of two ormore amino acid or nucleotide sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same, when compared andaligned for maximum correspondence, as measured using a sequencecomparison algorithm or by visual inspection.

For sequence comparison to determine percent nucleotide or amino acididentity, typically one sequence acts as a reference sequence, to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters. Optimal alignment of sequences for comparison can beconducted using BLAST set to default parameters.

As used with reference to polypeptides, the term “wild-type” refers toany polypeptide having an amino acid sequence present in a polypeptidefrom a naturally occurring organism, regardless of the source of themolecule; i.e., the term “wild-type” refers to sequence characteristics,regardless of whether the molecule is purified from a natural source;expressed recombinantly, followed by purification; or synthesized.

The term “mutation” shall mean a change in a protein, polypeptide, orpeptide sequence or subsequence produced by altering one or morenucleotides in a nucleotide coding for the protein, polypeptide, orpeptide, however the alteration is obtained. For example, a mutation canbe produced randomly, by PCR mutation, by synthesis of entire gene, orany other method.

The term “vector” is used herein to describe a DNA construct containinga polynucleotide. Such a vector can be propagated stably or transientlyin a host cell. The vector can, for example, be a plasmid, a viralvector, or simply a potential genomic insert. Once introduced into asuitable host, the vector may replicate and function independently ofthe host genome, or may, in some instances, integrate into the hostgenome.

As used herein, the terms “expression vector” or “expression construct”or “expression cassette” refer to a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. An “expression cassette” includes a codingnucleic acid (CDS) to be transcribed operably linked to a promoter and a3′UTR. Optionally, and in the Examples below, the promoter of anexpression cassette is a heterologous promoter.

“Exogenous gene” refers to a nucleic acid transformed into a cell. Theexogenous gene may be from a different species (and so heterologous), orfrom the same species (and so homologous) relative to the cell beingtransformed. In the case of a homologous gene, it occupies a differentlocation in the genome of the cell relative to the endogenous copy ofthe gene. The exogenous gene may be present in more than one copy in thecell. The exogenous gene may be maintained in a cell as an insertioninto the genome or as an episomal molecule.

An “inducible promoter” is one that mediates transcription of anoperably linked gene in response to a particular stimulus.

As used herein, the phrase “in operable linkage” refers to a functionallinkage between two sequences, such a control sequence (typically apromoter) and the linked sequence. A promoter is in operable linkagewith an exogenous gene if it can mediate transcription of the gene.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription.

As used herein, the term “recombinant” when used with reference, e.g.,to a cell, or nucleic acid, protein, or vector, indicates that the cell,nucleic acid, protein or vector, has been modified by the introductionof an exogenous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, over-expressed,under-expressed or not expressed at all. “Recombinant nucleic acid” asused herein refers to nucleic acid molecules that are initiallysynthesized through the use of laboratory methods, thereby creatingnucleic acid sequences that are not normally found in nature. By usinglaboratory methods, recombinant nucleic acid molecules in operablelinkage with different sequences (e.g., promoter, targeting sequence,etc.) is achieved. Thus an isolated nucleic acid, in a linear form, oran expression vector formed in vitro by ligating DNA molecules that arenot normally joined, are both considered recombinant for the purposes ofthis invention. It is understood that once a recombinant nucleic acid ismade and reintroduced into a host cell or organism, it will replicatenon-recombinantly, i.e., using the in vivo cellular machinery of thehost cell rather than in vitro manipulations; however, such nucleicacids, once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purposesherein. Similarly, a “recombinant protein” is a protein made usingrecombinant techniques, i.e., through the expression of a recombinantnucleic acid as depicted above.

A “transit peptide” is an amino acid sequence that directs thetrafficking of a polypeptide fused to the signal sequence. In connectionwith plastidic cells expressing the polypeptide, the transit peptide maydirect trafficking of the polypeptide to the plastid (i.e., a plastidtargeting peptide).

The term “polynucleotide” refers to a deoxyribonucleotide orribonucleotide polymer, and unless otherwise limited, includes knownanalogs of natural nucleotides that can function in a similar manner tonaturally occurring nucleotides. The term “polynucleotide” refers anyform of DNA or RNA, including, for example, genomic DNA; complementaryDNA (cDNA), which is a DNA representation of mRNA, usually obtained byreverse transcription of messenger RNA (mRNA) or amplification; DNAmolecules produced synthetically or by amplification; and mRNA. The term“polynucleotide” encompasses double-stranded nucleic acid molecules, aswell as single-stranded molecules. In double-stranded polynucleotides,the polynucleotide strands need not be coextensive (i.e., adouble-stranded polynucleotide need not be double-stranded along theentire length of both strands).

The term “host cell” refers to a cell capable of maintaining a vectoreither transiently or stably. Host cells include, without limitation,bacterial cells, yeast cells, insect cells, algal cells (e.g.,microalgal cells), plant cells and mammalian cells. Other host cellsknown in the art, or which become known, are also suitable for use inthe invention.

As used herein, the term “complementary” refers to the capacity forprecise pairing between two nucleotides. For example, if a nucleotide ata given position of a nucleic acid molecule is capable of hybridizingwith a nucleotide of another nucleic acid molecule, then the two nucleicacid molecules are considered to be complementary to one another at thatposition. The term “substantially complementary” describes sequencesthat are sufficiently complementary to one another to allow for specifichybridization under stringent hybridization conditions. In variousembodiments, the variant genes encoding variant FATB genes disclosedbelow can be replaced with a substantially complementary gene havingsuitable activity.

The phrase “stringent hybridization conditions” generally refers to atemperature about 5° C. lower than the melting temperature (Tm) for aspecific sequence at a defined ionic strength and pH. Exemplarystringent conditions suitable for achieving specific hybridization ofmost sequences are a temperature of at least about 60° C. and a saltconcentration of about 0.2 molar at pH 7.0.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a sequence alignment of the Cuphea hookeriana FATB2(SEQ ID NOS 69 and 70, respectively, in order of appearance) versus theCuphea avigera FATB1 (SEQ ID NOS 71 and 72, respectively, in order ofappearance) illustrates the two amino acid differences between thesethioesterases within their N-terminal specificity domain.

FIGS. 2A-B illustrate (A) a sequence alignment of FATB thioesterasesisolated from Cuphea genomes. The position of the conserved Methionerelative to the Catalytic Triad (Cys, His, and Asn) and N-terminalSpecificity domain is highlighted; and (B) a sequence comparison of theCpal FATB1, Ch FATB2 and Ca FATB1 surrounding the highlighted methione(SEQ ID NOS 73-75, respectively, in order of appearance). The Ca FATB1is unique due to the presence of a lysine instead of the methione.

FIGS. 3A-E illustrate histograms of C8-C14 fatty acid profiles ofmicroalgal oil with mean and median values for multiple transformants ofwild type and position 228 variant Cuphea hookeriana FATB2 (ChFATB2),Cuphea avigera FATB1 (CaFATB1) that depart from predictions based onprior data from an E. coli model.

DETAILED DESCRIPTION

Introduction

In illustrative embodiments, variant FATB acyl-ACP thioesterasesdescribed herein allow for control over acyl-ACP thioesterase substratespecificity. As a result, host cells expressing the acyl-ACPthioesterases produce oils with altered fatty acid profiles. In certainembodiments host cells expressing the variant acyl-ACP thioesterasesproduce triglceride-rich cell oils with fatty acid profilescharacterized by elevated mid chain fatty acids such as C8:0, C10:0,C12:0, and C14:0 fatty acids. A specific embodiment includes providing aFATB acyl-ACP thioesterase gene, mutating the gene so as to alter theamino acids in the gene product at the positions corresponding to H163and/or L186 of the reference Cuphea hookeriana FATB2 gene (SEQ ID NO:1). Optionally, the H163 and/or L186 mutant is combined with a mutationat M228.

As described in more detail in Example 1, by expressing such variantFATB2 genes, stably integrated in the nucleus of oleaginous plastidiccells, we produced strains that exceeded wildtype ChFATB2 expressingcontrol strains in terms of C8:0, C8:10 or the sum of C8:0 and C10:0production, including strains that produced oils with fatty acidprofiles where the C8 and C10 production exceed 9, 11, 14, or 18% of theprofile. In the latter case, the C8+C10 (i.e., the sum of C8:0 and C10:0production in the fatty acid profile as determined by FAME-GC with FIDdetection) level was more than doubled relative to the approximately 8%C8+C10 of the wildtype ChFATb2 strain. Specific variants with improvedC8+C10 production include those with P, K, or A at the 186 position; Yor F at the 163 position, or combinations thereof such as 186P/163Y,186P/163F, 186K/163Y, 186K/163F, 186A/163Y or 186A/163F. Of the doublemutants, we found that the H163Y/L186P variant produced an oil havingparticularly high concentrations of C8+C10. Using single or doublevariants, the C8:0 fatty acid profile percentages can be increased by50, 60, 70, 80, 100% or more relative to a control strain expressingwildtype ChFATB2; e.g. to more than 2, 2.5, 3, or 3.5% of the fatty acidprofile vs. 1.5% for the control (see Example 1).

The double mutants listed above can also be combined with a thirdmutation corresponding to 230 of Cuphea palustris FATB1. For many FATBgenes such as Cuphea hookeriana FATB2 and Cuphea avigera FATB1, thisresidue corresponds to residue 228. For example, an M228K mutation inCuphea hookeriana FATB2 expressed in an oleaginous eukaryotic microalgaincreased the C8/C10 ratio in the fatty acid profile of the oil fromabout 0.25 to about 1.0. Mutations at this position to Iso, Val, Phe,and Leu, Ala, or Thr in combination with the single or double mutants atpositions 186 and 163 discussed above, can also be advantageous.

Although Cuphea hookeriana FATB2 was used as a model system, the methodsof making the above-discussed mutations, methods of expressing these inan oleaginous cell, and methods of producing oil with these variants canbe applied to any acyl-ACP thioesterase gene, including those having atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity to SEQ ID NO:1, or the fragment of SEQ ID NO: 1 lacking thetransit peptide.

Although these variant genes were discovered using a eukaryoticmicroalgal expression system, the genes are more generally useful inways that are known in the art, including their expression in higherplants to produced altered triglyceride oils. When incorporated into anoleaginous cell (e.g., of an oilseed plant, algae (e.g., microalgae))the variant thioesterases can alter the fatty acid profiles of the cellto produce novel or more economical high-value commercial products.

The single, double or triple mutants can be used to produce an oil witha high ratio of C8:0 to C10:0 fatty acids. For example, the C8/10 ratiocan be equal to or greater than 0.3, 0.5, 0.7, 0.9, 1.0, 1.2, 1.4, 1.6,1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3.0.

The embodiments also encompass the residual biomass from such cellsafter oil extraction, oleochemicals, fuels and food products made fromthe oils and methods of cultivating the cells. In varying embodiments,the cells are microalgal cells, including heterotrophic or obligateheterotrophic cells, and cells classified as Chlorophyta,Trebouxiophyceae, Chlorellales, Chlorellaceae, or Chlorophyceae. Thecells can also be plant cells or cells of macroalgae. Host cells havinga type II fatty acid synthesis pathway are preferred. Although theexamples given below use the Trebouxiophyte Prototheca moriformis as ahost cell, the genes, constructs and methods disclosed may also find usein oilseed crops. Methods for introducing these genes into such cropssuch as soybean, corn, rapeseed, safflower, sunflower and others areknown in the art; see, for example, U.S. Pat. Nos. 6,331,664, 5,512,482,5,455,167, 5,667,997. Examples of oleochemicals include surfactants andsolvents made from fatty acids or oils.

Accordingly, in an embodiment, provided is a non-natural protein, anisolated gene encoding the non-natural protein, an expression cassetteexpressing the non-natural protein, or a host cell comprising theexpression cassette, wherein the non-natural protein has at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQID NO: 1 and comprises Y or F at the position corresponding to position163 of SEQ ID NO: 1 and/or P, K, or A at the position corresponding toposition 186 of SEQ ID NO: 1, and optionally K at the positioncorresponding to position 228 of SEQ ID NO: 1.

In a related embodiment, there is a method for producing a triglycerideoil. The method includes expressing, in a host cell, the protein ofmentioned immediately above, or a protein comprising at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity one of SEQID NOs: 3-8 that has Y or F at the position corresponding to position163 of SEQ ID NO: 1 and/or P, K, or A at the position corresponding toposition 186 of SEQ ID NO: 1, and optionally K at the positioncorresponding to position 228 of SEQ ID NO: 1. The method furtherincludes cultivating the host cell and isolating the oil.

In another embodiment, provided is a method for increasing the C8 and/orC10 fatty acids in a fatty acid profile of an oil produced by anoptionally oleaginous host cell. The method includes, providing a parentgene encoding a FATB enzyme, mutating the gene to so as to have Y or Fat the position corresponding to position 163 of SEQ ID NO: 1 and/or P,K, or A at the position corresponding to position 186 of SEQ ID NO: 1,and optionally K at the position corresponding to position 228 of SEQ IDNO: 1. The method further includes expressing the mutated gene in thehost cell and producing the oil. The fatty acid profile of the oil isthereby increased in C8 and/or C10 fatty acids relative to the parentgene. Optionally, the gene encoding the FATB enzyme encodes a proteinwith at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identity to SEQ ID NO: 1, 13 or 14.

As detailed in Example 3, compared to prior art work in E. Coli, thediscovery of the advantage of using Ala, or Thr at position 230 of CpalFATB1 (SEQ ID NO: 13) of in terms of C8+C10 production and/or increasedC8/C10 ratio, is new and unexpected. These novel mutations are usefulalone, in combination with a mutation at position 163 including theC8-favoring mutations disclosed herein, in combination with a mutationat position 186 including the C8-favoring mutations disclosed herein, orin combination with a double mutation at positions 163 and 186 includingthe C8-favoring mutations disclosed herein. Accordingly, in anembodiment, there is a non-natural protein, an isolated gene encodingthe non-natural protein, an expression cassette expressing thenon-natural protein, or a host cell comprising the expression cassette,wherein the non-natural protein has at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13 and A or Kat the position corresponding to position 230 of SEQ ID NO: 13. A methodfor producing an oil includes expressing, in a host cell, thenon-natural proteins described herein, cultivating the cell, andisolating the oil.

Variant Acyl-ACP Thioesterases

The variant TEs can be used in genetic constructs and geneticallyengineered oleaginous cells (e.g., plants, algae, microalgae) with oneor more exogenous genes to produce fatty acids, acylglycerides, orderivatives thereof. For example, microalgae or oilseed crops that wouldnaturally, or through genetic modification, produce high levels oflipids can be engineered (or further engineered) to express an exogenousvariant fatty acyl-ACP thioesterase, which can facilitate the cleavageof fatty acids from acyl carrier protein (ACP) during fatty acidsynthesis. The fatty acids synthesized may be incorporated into acylglycerides including triacylglycerides (TAGs, triglycerides). The TAGscan be recovered or, through further enzymatic processing within thecell, or in vitro, yield other useful compounds.

In an embodiment, the variant fatty acyl-ACP thioesterases are designedbased on the desired specificity for a growing (during fatty acidsynthesis) fatty acyl group having a particular carbon chain length. Aspecificity domain is selected based on its preference for a particularfatty acyl ACP substrate and/or for its ability to influence, increaseand/or promote the production of fatty acids of a desired carbon chainlength. Generally, the variant fatty acyl-ACP thioesterases havepreferential substrate specificity for mid-chain ACP-fatty acylsubstrates (e.g., to liberate C8, C10, C12, and/or C14 fatty acids). Invarying embodiments, the specificity domain in the N-terminus of theacyl-ACP thioesterase is heterologous (e.g., due to point mutationsand/or domain swapping) to the C-terminal catalytic domain. In certainembodiments, the fatty acid chain length substrate specificity and/orpreference of the specificity domain and the catalytic domain is thesame or within 1-2 carbons. For example, in varying embodiments, thevariant acyl-acyl carrier protein (ACP) thioesterase (TE) comprises:

Codon-Optimization for Expression

DNA encoding a polypeptide to be expressed in a microorganism, e.g., avariant acyl-ACP thioesterase and selectable marker can becodon-optimized cDNA. Methods of recoding genes for expression inmicroalgae are described in U.S. Pat. No. 7,135,290. Additionalinformation for codon optimization is available, e.g., at the CodonUsage Database at kazusa.or.jp/codon/. The table for Protothecapreferred codon usage is also provided in U.S. Patent Publ. No.2012/0283460, Table 1 of which is hereby incorporated herein byreference.

Expression and Targeting to Plastids

Proteins expressed in the nuclear genome of Prototheca can be targetedto the plastid using plastid targeting signals. Plastid targetingsequences endogenous to Chlorella are known, such as genes in theChlorella nuclear genome that encode proteins that are targeted to theplastid; see for example GenBank Accession numbers AY646197 andAF499684, and in one embodiment, such control sequences are used in thevectors described herein, e.g., to target expression of a protein to aPrototheca plastid.

The Examples below describe the use of algal plastid targeting sequencesto target heterologous proteins to the correct compartment in the hostcell. cDNA libraries were made using Prototheca moriformis and Chlorellaprotothecodies cells and are described in the Examples of U.S. PatentPubl. No. 2012/0283460 and in PCT Application No. PCT/US2009/066142.Amino acid sequences of the algal plastid targeting sequences identifiedfrom the cDNA libraries useful plastid targeting of recombinantlyexpressed variant acyl-ACP thioesterases are provided in U.S. PatentPubl. No. 2012/0283460 and herein. In varying embodiments, the plastidtransit peptide comprises an amino acid sequence selected from the groupconsisting of

(SEQ ID NO: 58) MATASTFSAFNARCGDLRRSAGSG PRRPARPLPVRGRA, (SEQ ID NO: 59)SGPRRPARPLPVR,  (SEQ ID NO: 60) SGPRRPARPLPVRAAIASEVPVATTSPR,(SEQ ID NO: 61) RPARPLPVRGRA,  (SEQ ID NO: 62) RPARPLPVRAAIASEVPVATTSPR,(SEQ ID NO: 63) RCGDLRRSAGSGPRRPARPLPVRGRA, (SEQ ID NO: 64)RCGDLRRSAGSGPRRPARPLPVRA  AIASEVPVATTSPR, (SEQ ID NO: 65) PARPLPVR,(SEQ ID NO: 66) PARPLPVRAAIASEVPVATTSPR,  (SEQ ID NO: 67) RRPARPLPVR,and (SEQ ID NO: 68) RRPARPLPVRAAIASEVPVATTSPR.

Where novel FATB variants are disclosed here, it will be understood thata variety of heterologous plastid transit peptides can be used. In otherwords, the non-targeting peptide domain is more highly conserved.Accordingly, embodiments described herein feature the novel FATBenzymatic domain with or without a plastid targeting sequence. Forexample, where a percent identity to a novel FATB gene is given herein,the same identity can be applied (where specified) to the same sequenceabsent the targeting peptide. A substitute targeting peptide canoptionally be used in connection with such a sequence.

Host Cells—Oil- or Lipid-Producing Microorganisms

Any species of organism that produces suitable lipid and/or hydrocarboncan be used, although microorganisms that naturally produce high levelsof suitable lipid and/or hydrocarbon are preferred. Production ofhydrocarbons by microorganisms is reviewed by Metzger et al. ApplMicrobiol Biotechnol (2005) 66: 486-496 and A Look Back at the U.S.Department of Energy's Aquatic Species Program: Biodiesel from Algae,NREUTP-580-24190, John Sheehan, Terri Dunahay, John Benemann and PaulRoessler (1998).

Considerations for the selection of microorganisms include, in additionto production of suitable lipids or hydrocarbons for production of oils,fuels, and oleochemicals: (1) high lipid content as a percentage of cellweight; (2) ease of growth; (3) ease of genetic engineering; and (4)ease of biomass processing. In particular embodiments, the wild-type orgenetically engineered microorganism yields cells that are at least 40%,at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, orat least 70% or more lipid. Preferred organisms grow heterotrophically(on sugars in the absence of light) or can be engineered to do so using,for example, methods disclosed herein. The ease of transformation andavailability of selectable markers and promoters, constitutive orinducible, that are functional in the microorganism affect the ease ofgenetic engineering. Processing considerations can include, for example,the availability of effective means for lysing the cells.

A. Algae

In one embodiment, the microorganism is a microalgae. Nonlimitingexamples of microalgae that can be used for expression of variantacyl-ACP thioestesterases include, e.g., Achnanthes orientalis,Agmenellum, Amphiprora hyaline, Amphora coffeiformis, Amphoracoffeiformis linea, Amphora coffeiformis punctata, Amphora coffeiformistaylori, Amphora coffeiformis tenuis, Amphora delicatissima, Amphoradelicatissima capitata, Amphora sp., Anabaena, Ankistrodesmus,Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp.,Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor,Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis,Chaetoceros muelleri, Chaetoceros muelleri subsalsum, Chaetoceros sp.,Chlorella anitrata, Chlorella Antarctica, Chlorella aureoviridis,Chlorella candida, Chlorella capsulate, Chlorella desiccate, Chlorellaellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var.vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorellainfusionum var. actophila, Chlorella infusionum var. auxenophila,Chlorella kessleri, Chlorella lobophora (strain SAG 37.88), Chlorellaluteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorellaluteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima,Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorellaparva, Chlorella photophila, Chlorella pringsheimii, Chlorellaprotothecoides (including any of UTEX strains 1806, 411, 264, 256, 255,250, 249, 31, 29, 25), Chlorella protothecoides var. acidicola,Chlorella regularis, Chlorella regularis var. minima, Chlorellaregularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila,Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorellasimplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica,Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris,Chlorella vulgaris f. tertia, Chlorella vulgaris var. autotrophica,Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris,Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var.vulgaris f. viridis, Chlorella xanthella, Chlorella zofingiensis,Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum,Chlorococcum sp., Chlorogonium, Chroomonas sp., Chrysosphaera sp.,Cricosphaera sp., Crypthecodinium cohnii, Cryptomonas sp., Cyclotellacryptica, Cyclotella meneghiniana, Cyclotella sp., Dunaliella sp.,Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate,Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliellapeircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola,Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta,Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena,Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp.,Gloeothamnion sp., Hymenomonas sp., Isochrysis aff galbana, Isochrysisgalbana, Lepocinclis, Micractinium, Micractinium (UTEX LB 2614),Monoraphidium minutum, Monoraphidium sp., Nannochloris sp.,Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata,Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa,Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp.,Nitschia communis, Nitzschia alexandrina, Nitzschia communis, Nitzschiadissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschiainconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschiapusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis,Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva,Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoriasp., Oscillatoria subbrevis, ParaChlorella kessleri, Pascheriaacidophila, Pavlova sp., Phagus, Phormidium, Platymonas sp.,Pleurochrysis carterae, Pleurochrysis dentate, Pleurochrysis sp.,Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis,Prototheca moriformis, Prototheca zopfii, Pseudo Chlorella aquatica,Pyramimonas sp., Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte,Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis,Stichococcus sp., Synechococcus sp., Tetraedron, Tetraselmis sp.,Tetraselmis suecica, Thalassiosira weissflogii, and Viridiellafridericiana.

Illustrative host cells feature oleaginous cells that produce alteredfatty acid profiles and/or altered regiospecific distribution of fattyacids in glycerolipids, and products produced from the cells. Examplesof oleaginous cells include microbial cells having a type II lipidbiosynthesis pathway, including plastidic oleaginous cells such as thoseof oleaginous algae. Specific examples of cells include heterotrophic orobligate eukaryotic heterotophic microalgae of the phylum Chlorpophya,the class Trebouxiophytae, the order Chlorellales, or the familyChlorellacae. Examples of oleaginous microalgae are provided inPublished PCT Patent Applications WO2008/151149, WO2010/06032,WO2011/150410, and WO2011/150411, including species of Chlorella andPrototheca, a genus comprising obligate heterotrophs. The oleaginouscells can be, for example, capable of producing 25%, 30%, 40%, 50%, 60%,70%, 80%, 85%, or about 90% oil by cell weight, ±5%. The above mentionedpublications also disclose methods for cultivating such cells andextracting oil, especially from microalgal cells; such methods areapplicable to the cells disclosed herein. In any of the embodimentsdescribed herein, the cells can be heterotrophic cells comprising anexogenous invertase gene so as to allow the cells to produce oil from asucrose feedstock.

Illustrative embodiments of host cells include recombinant oleaginouscells expressing one or more exogenous genes encoding fatty acidbiosynthesis enzymes. As a result, some embodiments feature cell oilsnever before obtainable in a cell oil. In some cases, the cell oils werenot obtainable from a non-plant or non-seed oil, or not obtainable atall.

The oleaginous cells produce a storage oil, which may be stored instorage vesicles of the cell. A raw cell oil may be obtained from thecells by disrupting the cells and isolating the oil. The oils producedmay be refined, bleached and deodorized (RBD) as known in the art or asdescribed in WO2010/120939. The raw or RBD oils may be used in a varietyof food, chemical, and industrial products or processes. After recoveryof the oil, a valuable residual biomass remains. Uses for the residualbiomass include the production of paper, plastics, absorbents,adsorbents, as animal feed, for human nutrition, or for fertilizer.

Where a fatty acid profile of a triglyceride cell oil is given, it willbe understood that this refers to a nonfractionated sample of thestorage oil extracted from the cell analyzed under conditions in whichphospholipids have been removed or with an analysis method that issubstantially insensitive to the fatty acids of the phospholipids (e.g.using chromatography and mass spectrometry). Because the cells areoleaginous, in some cases the storage oil will constitute the bulk ofall the TAGs in the cell.

In varying embodiments, the host cell is a plastidic cell, e.g., aheterotrophic microalgae of the phylum Chlorpophya, the classTrebouxiophytae, the order Chlorellales, or the family Chlorellacae. Invarying embodiments, the cell is oleaginous and capable of accumulatingat least 40% oil by dry cell weight. The cell can be an obligateheterotroph, such as a species of Prototheca, including Protothecamoriformis or Prototheca zopfii. The nucleic acid encoding the variantacyl-ACP TEs described herein can also be expressed in autotrophic algaeor plants. Optionally, the cell is capable of using sucrose to produceoil and a recombinant invertase gene may be introduced to allowmetabolism of sucrose, as described in PCT Publications WO2008/151149,WO2010/06032, WO2011/150410, WO2011/150411, and international patentapplication PCT/US12/23696. The invertase may be codon optimized andintegrated into a chromosome of the cell, as may all of the genesmentioned here. Codon usage for different algal and plant species ofinterest is known in the art and can be found, e.g., on the internet atthe Codon Usage Database at kazusa.or.jp/codon/.

The polynucleotides encoding the variant acyl-ACP TEs described hereinfurther can be expressed in a wide variety of plant host cells. Ofparticular interest are plant cells of plants involved in the productionof vegetable oils for edible and industrial uses, including e.g.,temperate oilseed crops. Plants of interest include, but are not limitedto, grapeseed (Canola and High Erucic Acid varieties), sunflower,safflower, cotton, Cuphea, soybean, peanut, coconut and oil palms, andcorn. See, U.S. Pat. Nos. 5,850,022; 5,723,761; 5,639,790; 5,807,893;5,455,167; 5,654,495; 5,512,482; 5,298,421; 5,667,997; and 5,344,771;5,304,481.

Oils with Non-Naturally Occurring Fatty Acid Profiles

Oils disclosed herein are distinct from other naturally occurring oilsthat are high in mid-chain fatty acids, such as palm oil, palm kerneloil, and coconut oil. For example, levels of contaminants such ascarotenoids are far higher in palm oil and palm kernel oil than in theoils described herein. Palm and palm kernel oils in particular containalpha and beta carotenes and lycopene in much higher amounts than is inthe oils described herein. In addition, over 20 different carotenoidsare found in palm and palm kernel oil, whereas the Examples demonstratethat the oils described herein contain very few carotenoids species andvery low levels. In addition, the levels of vitamin E compounds such astocotrienols are far higher in palm, palm kernel, and coconut oil thanin the oils described herein.

Generally, Prototheca strains have very little or no fatty acids withthe chain length C8-C14. For example, Prototheca strains Protothecamoriformis (UTEX 1435), Prototheca krugani (UTEX 329), Protothecastagnora (UTEX 1442) and Prototheca zopfii (UTEX 1438) produce no (orundetectable amounts) C8 fatty acids, between 0-0.01% C10 fatty acids,between 0.03-2.1% C12 fatty acids and between 1.0-1.7% C14 fatty acids.

In some cases, the oleaginous cells (e.g, Prototheca strains) containinga transgene encoding a variant fatty acyl-ACP thioesterase has a fattyacid profile characterized by 5-10%, 10-20%, 20-30%, 30-40%, 40-50%,50-60%, 60-70%, 70-80%, 80-90%, or 90-99% C8, C10, C12, or C14 fattyacids. In other cases, the Prototheca strains containing a transgeneencoding a fatty acyl-ACP thioesterase that has activity towards fattyacyl-ACP substrates of chain length C12 and C14 and produces fatty acidsof the chain length C12 and the chain length C14 at a ratio of1:1+/−20%.

In some instances, keeping the transgenic Prototheca strains underconstant and high selective pressure to retain exogenous genes isadvantageous due to the increase in the desired fatty acid of a specificchain length. High levels of exogenous gene retention can also beachieved by inserting exogenous genes into the nuclear chromosomes ofthe cells using homologous recombination vectors and methods disclosedherein. Recombinant cells containing exogenous genes integrated intonuclear chromosomes are also contemplated.

Microalgal oil can also include other constituents produced by themicroalgae, or incorporated into the microalgal oil from the culturemedium. These other constituents can be present in varying amountdepending on the culture conditions used to culture the microalgae, thespecies of microalgae, the extraction method used to recover microalgaloil from the biomass and other factors that may affect microalgal oilcomposition. Non-limiting examples of such constituents includecarotenoids, present from 0.1-0.4 micrograms/ml, chlorophyll presentfrom 0-0.02 milligrams/kilogram of oil, gamma tocopherol present from0.4-0.6 milligrams/100 grams of oil, and total tocotrienols present from0.2-0.5 milligrams/gram of oil.

The other constituents can include, without limitation, phospholipids,tocopherols, tocotrienols, carotenoids (e.g., alpha-carotene,beta-carotene, lycopene, etc.), xanthophylls (e.g., lutein, zeaxanthin,alpha-cryptoxanthin and beta-crytoxanthin), and various organic orinorganic compounds.

In some cases, the oil extracted from Prototheca species comprises nomore than 0.02 mg/kg chlorophyll. In some cases, the oil extracted fromPrototheca species comprises no more than 0.4 mcg/ml total carotenoids.In some cases the Prototheca oil comprises between 0.40-0.60 milligramsof gamma tocopherol per 100 grams of oil. In other cases, the Protothecaoil comprises between 0.2-0.5 milligrams of total tocotrienols per gramof oil.

Oils produced from host cells expressing a variant acyl-ACP thioesterasewill have an isotopic profile that distinguishes it, e.g., from blendedoils from other sources. The stable carbon isotope value δ13C is anexpression of the ratio of 13C/12C relative to a standard (e.g. PDB,carbonite of fossil skeleton of Belemnite americana from Peedeeformation of South Carolina). The stable carbon isotope value δ13C(0/00) of the oils can be related to the δ13C value of the feedstockused. In some embodiments the oils are derived from oleaginous organismsheterotrophically grown on sugar derived from a C4 plant such as corn orsugarcane. In some embodiments, the δ13C (0/00) of the oil is from 10 to−17 0/00 or from 13 to −16 0/00.

In varying embodiments, a host cell expressing a variant acyl-ACPthioesterase comprising all or specificity-determining residues of aspecificity domain from a C10-preferring acyl-ACP thioesterase (e.g., anacyl-ACP thioesterase from Cuphea hookeriana), and a catalytic domainfrom a C12-preferring acyl-ACP thioesterase (e.g., an acyl-ACPthioesterase from Cuphea wrightii or Umbellularia californica) producesan oil comprising at least about 10% C12:0 fatty acids, and at leastabout 10% C14:0 fatty acids.

In varying embodiments, a host cell expressing a variant acyl-ACPthioesterase comprising all or specificity-determining residues of amodified specificity domain of a first acyl-ACP thioesterase having oneor both His163→Tyr or Leu186→Pro substitutions (or at positionscorresponding to His163→Tyr or Leu186→Pro of SEQ ID NO:61), and acatalytic domain of a second acyl-ACP thioesterase produces an oilcomprising at least about 5%, e.g., at least about 6%, 7%, 8%, 9%, 10%,12%, 15%, or more, C8:0 fatty acids or at least about 5%, e.g., at leastabout 6%, 7%, 8%, 9%, 10%, 12%, 15%, or more, C10:0 fatty acids or aC8:0/C10:0 ratio that is at least about 5%, e.g., at least about 6%, 7%,8%, 9%, 10%, 12%, 15%, or more. As appropriate, the specificity domaincan be derived from a C8:0-, C10:0- or a C12:0-preferring acyl-ACPthioesterase and independently the catalytic domain can be derived froma C8:0-, C10:0- or a C12:0-preferring acyl-ACP thioesterase. Thespecificity domain and the catalytic domain can be from the same ordifferent acyl-ACP thioesterases. In varying embodiments, a host cellexpressing a variant acyl-ACP thioesterase comprising all orspecificity-determining residues of a modified specificity domain from aC10-preferring acyl-ACP thioesterase (e.g., an acyl-ACP thioesterasefrom Cuphea hookeriana having one or both His163→Tyr or Leu186→Prosubstitutions), and a catalytic domain from a C10-preferring acyl-ACPthioesterase (e.g., an acyl-ACP thioesterase from Cuphea hookeriana)produces an oil comprising at least about 5%, e.g., at least about 6%,7%, 8%, 9%, 10%, 12%, 15%, or more, C8:0 fatty acids or at least about5%, e.g., at least about 6%, 7%, 8%, 9%, 10%, 12%, 15%, or more, C10:0fatty acids or a C8:0/C10:0 ratio that is at least about 5%, e.g., atleast about 6%, 7%, 8%, 9%, 10%, 12%, 15%, or more.

In varying embodiments, a host cell expressing a variant acyl-ACPthioesterase comprising all or specificity-determining residues of aspecificity domain from a C14-preferring acyl-ACP thioesterase (e.g., anacyl-ACP thioesterase from Cinnamomum camphorum), and a catalytic domainfrom a C12-preferring acyl-ACP thioesterase (e.g., an acyl-ACPthioesterase from Cuphea wrightii or Umbellularia californica) producesan oil comprising C12:0 fatty acids and C14:0 fatty acid at anapproximate 1:1 ratio; e.g, a ratio of 1:1+/−20%.

Further, host cells expressing a variant acyl-ACP thioesterasecomprising 5 or more amino acid residues extending from the C-terminusof a linker domain positioned N-terminal to the hydrophobic domain,produce an oil comprising relatively elevated mid-chain length fattyacids (e.g., C8:0, C10:0, C12:0, C14:0) in comparison to host cellsexpressing the same acyl-ACP thioesterase without a linker domain. Invarying embodiments, host cells expressing a variant acyl-ACPthioesterase comprising 5 or more amino acid residues extending from theC-terminus of a linker domain positioned N-terminal to the hydrophobicdomain, produce an oil comprising mid-chain length fatty acids increasedby at least 1-fold, 2-fold, 3-fold, or more, in comparison to host cellsexpressing the same acyl-ACP thioesterase without a linker domain.

In a specific embodiment, a recombinant cell comprises nucleic acidsoperable to express a product of an exogenous gene encoding a variantacyl-ACP thioesterase exogenous gene encoding an active acyl-ACPthioesterase that catalyzes the cleavage of mid-chain fatty acids fromACP. As a result, in one embodiment, the oil produced can becharacterized by a fatty acid profile elevated in C8, C10, C12, and/orC14 fatty acids and reduced in C16, C18, and C18:1 fatty acids as aresult of expression of the recombinant nucleic acids. In varyingembodiments, the increase in C8, C10, C12, and/or C14 fatty acids isgreater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%,50%, 60%, 70%, from 75-85%, from 70-90%, from 90-200%, from 200-300%,from 300-400%, from 400-500%, or greater than 500%.

In some embodiments, an additional genetic modification to increase thelevel of mid-chain fatty acids in the cell or oil of the cell includesthe expression of an exogenous lysophosphatidic acid acyltransferasegene encoding an active lysophosphatidic acid acyltransferase (LPAAT)that catalyzes the transfer of a mid-chain fatty-acyl group to the sn-2position of a substituted acylglyceroester. In a specific relatedembodiment, both an exogenous acyl-ACP thioesterase and LPAAT are stablyexpressed in the cell. As a result of introducing recombinant nucleicacids into an oleaginous cell (and especially into a plastidic microbialcell) an exogenous mid-chain-specific thioesterase and an exogenousLPAAT that catalyzes the transfer of a mid-chain fatty-acyl group to thesn-2 position of a substituted acylglyceroester, the cell can be made toincrease the percent of a particular mid-chain fatty acid in thetriacylglycerides (TAGs) that it produces by 10, 20 30, 40, 50, 60, 70,80, 90-fold, or more. Introduction of the exogenous LPAAT can increasemid-chain fatty acids at the sn-2 position by 1, 2, 3, 4 fold or morecompared to introducing an exogenous mid-chain preferring acyl-ACPthioesterase alone. In an embodiment, the mid-chain fatty acid isgreater than 30, 40, 50 60, 70, 80, or 90% of the TAG fatty acidsproduced by the cell. In various embodiments, the mid-chain fatty acidis capric, caprylic, lauric, myristic, and/or palmitic.

In varying embodiments, the gene encoding an lysophosphatidic acidacyltransferase (LPAAT) is selected from the group consisting ofArabidopsis thaliana 1-acyl-sn-glycerol-3-phosphate acyltransferase(GenBank Accession No. AEE85783), Brassica juncea1-acyl-sn-glycerol-3-phosphate acyltransferase (GenBank Accession No.ABQ42862), Brassica juncea 1-acyl-sn-glycerol-3-phosphateacyltransferase (GenBank Accession No. ABM92334), Brassica napus1-acyl-sn-glycerol-3-phosphate acyltransferase (GenBank Accession No.CAB09138), Chlamydomonas reinhardtii lysophosphatidic acidacyltransferase (GenBank Accession No. EDP02300), Cocos nuciferalysophosphatidic acid acyltransferase (GenBank Acc. No. AAC49119),Limnanthes alba lysophosphatidic acid acyltransferase (GenBank AccessionNo. EDP02300), Limnanthes douglasii 1-acyl-sn-glycerol-3-phosphateacyltransferase (putative) (GenBank Accession No. CAA88620), Limnanthesdouglasii acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase(GenBank Accession No. ABD62751), Limnanthes douglasii1-acylglycerol-3-phosphate O-acyltransferase (GenBank Accession No.CAA58239), Ricinus communis 1-acyl-sn-glycerol-3-phosphateacyltransferase (GenBank Accession No. EEF39377).

Alternately, or in addition to expression of an exogenous LPAAT, thecell may comprise recombinant nucleic acids that are operable to expressan exogenous KASI or KASIV enzyme and optionally to decrease oreliminate the activity of a KASII, which is particularly advantageouswhen a mid-chain-preferring acyl-ACP thioesterase is expressed.Engineering of Prototheca cells to overexpress KASI and/or KASIV enzymesin conjunction with a mid-chain preferring acyl-ACP thioesterase cangenerate strains in which production of C10-C12 fatty acids is at leastabout 40% of total fatty acids, e.g., at least about 45%, 50%, 55%, 60%or more, of total fatty acids. Mid-chain production can also beincreased by suppressing the activity of KASI and/or KASII (e.g., usinga knockout or knockdown). Chromosomal knockout of different alleles ofPrototheca moriformis (UTEX 1435) KASI in conjunction withoverexpression of a mid-chain preferring acyl-ACP thioesterase canachieve fatty acid profiles that are at least about 60% C10-C14 fattyacids, e.g., at least about 65%, 70%, 75%, 80%, 85% or more C10-C14fatty acids. Elevated mid-chain fatty acids can also be achieved as aresult of expression of KASI RNA hairpin polynucleotides. In addition toany of these modifications, unsaturated or polyunsaturated fatty acidproduction can be suppressed (e.g., by knockout or knockdown) of a SADor FAD enzyme.

In an embodiment, one of the above described high mid-chain producingcells is further engineered to produce a low polyunsaturated oil byknocking out or knocking down one or more fatty acyl desaturases.Accordingly, the oil produced has high stability.

The high mid-chain oils or fatty acids derived from hydrolysis of theseoils may be particularly useful in food, fuel and oleochemicalapplications including the production of lubricants and surfactants. Forexample, fatty acids derived from the cells can be esterified, cracked,reduced to an aldehyde or alcohol, aminated, sulfated, sulfonated, orsubjected to other chemical process known in the art.

The invention, having been described in detail above, is exemplified inthe following examples, which are offered to illustrate, but not tolimit, the claimed invention.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1: Mutagenesis of Cuphea hookeriana FATB2

We modified the activity and specificity of a FATB2 thioesteraseoriginally isolated from Cuphea hookeriana (Ch FATB2, accession U39834),using site directed mutagenesis of H163 and L186 within the enzymaticcore (H163 and L186 within Ch FATB2.).

For the above examples, an expression construct was used that targetedthe FATB variants and selection markers to the Thi4 (thiaminebiosynthesis) locus. An antibiotic resistance gene was used to selectfor resistance to G418 antibiotic. The UAPA promoter was used to driveFATB. The construct is exemplified in SEQ ID NO: 9.

As disclosed in PCT/US2014/013676 we discovered that grafting the Cupheaavigera FATB1 (Ca FATB1) N-terminal specificity domain (FIG. 2B) ontothe Cuphea hookeriana FATB2 (FIG. 2A) improves activity and C8-C10ratio. Prototheca moriformis transformants expressing Ch FATB2 H163Y,L186P (D3130) mutants exhibited about 2 fold increase in the averageC8-C10 sum as well as a shift in fatty acid profile specificity relativeto the wild-type Ch FATB2 (D3042).

The His at position 163 within the Ch FATB2 (FIG. 2A) is highlyconserved across FATB thioesterases. In contrast, the Leu at position186 within the Ch FATB is rare. In other FATB's, position 186 istypically occupied by a Pro or Leu. Due to these observations and alsothe increased activity and shift in fatty acid profile specificity ofPrototheca moriformis strains expressing the Ch FATB2 H163Y, L186Pmutant (D3130), we identified H163 and L186 as “hot spots” formutagenesis and performed exhaustive mutagenesis at both H163 and L186to explore the effect of amino acid combinations on activity of the ChFATB2 when expressed within the Prototheca moriformis model system.Details of the cloning system are given in PCT/US2014/013676.

Thirty-eight individual Ch FATB2 variants were generated and theireffect on C8:0 and C10:0 fatty acid accumulation was quantified.Transformants with C8-C10 sum within 3 standard deviations above thewild-type Ch FATB2 control (D3598) were classified as positive and thosewithin 3 standard deviations below were scored as negative. See Table 1.The remaining transformants were classified as neutral. As shown inTable 1, Prototheca moriformis transformed with six of the Ch FATB2mutants (D3570, D3573, D3582, D3584, D3588, and D3599) accumulatedC8:0-C10:0 fatty acids within 3 standard above transformants expressingthe wild type Ch FATB2 (D3598) control.

TABLE 1 Analysis of FATB varaiants for C8-C14 fatty acid productionin P.moriformis.* Ch FABT2 variant C8:0 C10:0 C12:0 C14:0 C8-C10sumD3565-186V average 1.82 6.86 0.20 1.64 8.68 STDEV 0.28 0.76 0.02 0.051.04 D3566-186Y average 1.94 7.17 0.22 1.72 9.11 STDEV 0.45 1.11 0.030.08 1.56 D3567-186W average 0.78 3.98 0.16 1.66 4.76 (negative) STDEV0.06 0.20 0.05 0.02 0.26 D3568-186T average 1.78 7.00 0.21 1.61 8.78STDEV 0.38 0.89 0.02 0.06 1.27 D3569-186S average 1.85 7.05 0.20 1.618.9 STDEV 0.32 0.75 0.02 0.06 1.07 D3570-186P average 2.86 8.68 0.251.65 11.54 (positive) STDEV 0.25 0.61 0.02 0.05 0.86 D3571-186F average1.46 5.98 0.19 1.62 7.44 STDEV 0.32 0.78 0.02 0.06 1.1 D3572-186Maverage 1.48 6.13 0.18 1.59 7.61 STDEV 0.23 0.46 0.01 0.03 0.69D3573-186K average 2.62 8.74 0.23 1.54 11.36 (positive) STDEV 0.66 1.330.03 0.05 1.99 D3574-186I average 1.56 6.35 0.18 1.63 7.91 STDEV 0.260.55 0.01 0.04 0.81 D3575-186H average 1.97 7.38 0.19 1.60 9.35 STDEV0.29 0.58 0.01 0.03 0.87 D3576-186G average 1.41 5.83 0.18 1.70 7.24STDEV 0.25 0.69 0.02 0.07 0.94 D3577-186E average 2.14 7.91 0.21 1.6810.05 STDEV 0.54 1.29 0.03 0.08 1.83 D3578-186Q average 2.07 7.69 0.201.61 9.76 STDEV 0.46 0.96 0.02 0.06 1.42 D3579-186C average 0.95 4.770.16 1.60 5.72 STDEV 0.07 0.23 0.01 0.03 0.3 D3580-186N average 2.257.93 0.21 1.55 10.18 STDEV 0.33 0.79 0.02 0.04 1.12 D3581-186R average2.21 7.74 0.22 1.63 9.95 STDEV 0.73 1.90 0.04 0.07 2.63 D3582-186Aaverage 2.39 8.74 0.23 1.58 11.13 (positive) STDEV 0.78 1.94 0.04 0.062.72 D3603-186D average 1.91 7.02 0.19 1.54 8.93 STDEV 0.41 1.14 0.020.05 1.55 D3583-163V average 0.00 0.12 0.03 1.89 0.12 (negative) STDEV0.00 0.07 0.04 0.21 0.07 D3584-163Y average 3.71 10.52 0.30 1.61 14.23(positive) STDEV 0.92 1.75 0.04 0.04 2.67 D3585-163W average 1.11 4.880.18 1.67 5.99 STDEV 0.12 0.28 0.01 0.04 0.4 D3586-163T average 0.000.01 0.01 1.78 0.01 (negative) STDEV 0.00 0.03 0.02 0.13 0.03 D3587-163Paverage 0.00 0.01 0.01 1.84 0.01 (negative) STDEV 0.00 0.03 0.03 0.140.03 D3588-163F average 3.79 10.82 0.31 1.59 14.61 (positive) STDEV 0.540.77 0.01 0.03 1.31 D3589-163K average 0.00 0.01 0.06 1.79 0.01(negative) STDEV 0.00 0.02 0.01 0.07 0.02 D3590-163L average 1.95 7.490.20 1.66 9.44 STDEV 0.38 1.15 0.03 0.08 1.53 D3591-163I average 0.060.70 0.07 1.74 0.76 (negative) STDEV 0.02 0.15 0.01 0.11 0.17 D3592-163Gaverage 0.00 0.01 0.06 1.81 0.01 (negative) STDEV 0.00 0.02 0.01 0.030.02 D3593-163E average 0.00 0.02 0.06 1.99 0.02 (negative) STDEV 0.010.05 0.02 0.20 0.06 D3594-163Q average 0.06 0.69 0.07 1.74 0.75(negative) STDEV 0.05 0.36 0.01 0.06 0.41 D3595-163C average 0.00 0.020.01 1.80 0.02 (negative) STDEV 0.00 0.05 0.02 0.17 0.05 D3596-163Raverage 0.00 0.01 0.01 1.92 0.01 (negative) STDEV 0.00 0.04 0.02 0.360.04 D3597-163A average 0.00 0.00 0.01 1.72 0 (negative) STDEV 0.00 0.000.03 0.14 0 D3600-163S12 average 0.00 0.00 0.02 1.74 0 (negative) STDEV0.00 0.00 0.03 0.12 0 D3601-163M average 0.02 0.76 0.02 1.75 0.78(negative) STDEV 0.05 0.16 0.04 0.15 0.21 D3602-163N average 0.00 0.000.01 1.74 0 (negative) STDEV 0.00 0.00 0.02 0.07 0 D3609-163D average0.00 0.00 0.01 1.80 0 (negative) STDEV 0.00 0.00 0.02 0.15 0 D3598- wildtype average 1.52 6.55 0.19 1.60 8.07 Ch FATB2 STDEV 0.19 0.62 0.02 0.110.81 D3599- H163Y, average 5.77 12.50 0.39 1.73 18.27 L186P(positive)STDEV 0.63 0.99 0.03 0.05 1.62 *12 transformants were screened permutant. The length of lipid production unde low lintrogen conditions was3 days.

In summary, we have shown that it is possible to increase activity andshift profile specificity within C8-C10 specific FATB thioesterasesderived from Cuphea hookeriana by using site directed mutagenesis ofH163 and L186 within the N-terminal specificity domain. We found cellsexpressing variants that exceeded the parent ChFATB2 sequence in termsof sum of C8:0+C10:0 production including strains that produced oilswith fatty acid profiles where the C8 and C10 production exceed 9, 11,14, of the profile.

Example 2: Identification of Double Mutants in FATB

Based on the demonstrated ability to modify the activity and specificityof a FATB2 thioesterase originally isolated from Cuphea hookeriana (ChFATB2, accession U39834), using site directed mutagenesis of H163 andL186 a second round of mutagenesis was initiated. Six constructscombining the positive mutations from Rd1 (C8+C10 within 3 standarddeviations above the wild-type Ch FATB2 control (D3598)) were generated(Table 2).

TABLE 2 Beneficial Mutations Constructs 1) 163Tyr 186Lys 2) 163Tyr186Ala 3) 163Phe 186Pro 4) 163Phe 186Lys 5) 163Phe 186Ala

For the above examples, an expression construct was used that targetedthe FATB variants and selection markers to the Thi4 (thiaminebiosynthesis) locus. An antibiotic resistance gene was used to selectfor resistance to G418 antibiotic. The UAPA promoter was used to driveFATB. The construct is exemplifided in SEQ ID NO: 9.

Five individual Ch FATB2 variants were generated and their effect onC8:0 and C10:0 fatty acid accumulation was quantified. Transformantswith C8-C10 sum within 3 standard deviations above the wild-type ChFATB2 control (D3598) were classified as positive (Table 3) and thosewithin 3 standard deviations below were scored as negative (Table 3).The remaining transformants were classified as neutral. As shown inTable 3, Prototheca moriformis transformed with three of the Ch FATB2mutants (D3875, D3876, and D3885) accumulated C8:0-C10:0 fatty acidswithin 3 standard above transformants expressing the wild type Ch FATB2(D3598) control.

TABLE 3 Ch FABT2 variant C8:0 C10:0 C12:0 C14:0 C8-C10sum D3875-163F,186A average 4.66 12.40 0.34 1.61 19.02 (positive) STDEV 1.27 2.39 0.050.19 3.73 D3876-163F,186K average 5.25 13.12 0.36 1.55 20.28 (positive)STDEV 1.19 2.05 0.05 0.03 3.31 D3877-163F, 186P average 0.00 0.00 0.001.90 1.90 (negative) STDEV 0.00 0.00 0.00 0.28 0.28 D3884-163Y, 186Aaverage 4.29 11.69 0.32 1.52 17.81 STDEV 0.58 1.06 0.03 0.04 1.69D3885-163Y, 186K average 5.39 13.14 0.36 1.49 20.38 (positive) STDEV1.31 2.18 0.05 0.03 3.52 D3598- wild type average 1.14 5.72 0.15 1.568.57 Ch FATB2 STDEV 0.27 0.77 0.06 0.05 1.12 D3599- H163Y, average 5.6512.91 0.39 1.74 20.69 L186P STDEV 1.29 2.06 0.06 0.02 3.42

Example 3: Mutations at FATB Position 230

In the example below, we demonstrate the ability to modify the activityand specificity of three FATB thioesterases originally isolated fromCuphea hookeriana (Ch FATB2, Uniprot accession U39834), Cuphea palustris(Cpal FATB1, Uniprot accession Q39554, SEQ ID NO: 13) and Cuphea avigeraFATB1 (Ca FATB1 accession R4J2L6, SEQ ID NO: 14) using site directedmutagenesis of a conserved Met within the enzymatic core (M230 withinCpal FATB1).

It has recently been reported that substitution of the conserved M230within the Cpal FATB1 with Iso, Val, Phe or Leu will increase theenzymatic activity of this thioesterase. Because these results wereobtained using E. coli, we performed a similar screen to see if theresults could be reproduced when expressed in Prototheca moriformismicroalgae. The wild-type and thirteen Cpal FATB1 M230 mutants weregenerated and their effect on C8:0 fatty acid accumulation quantified.As shown in Table 4, Prototheca moriformis transformed with six of theCpal FATB1 M230 mutants (D3206, D3208, D3211, D3212, D3214, and D3215)exhibited fatty acid profiles that were similar to the non-transformedS6165 host algal strain which likely is due to the mutation inactivatingthe Cpal FATB1 enzyme. In contrast, Prototheca moriformis transformantsexpressing one of the remaining seven Cpal FATB1 M230 mutantsaccumulated C8:0 fatty acids to varying degrees above thenon-transformed S6165 host. D3213 (M230P) was less effective than thewild-type Cpal FATB1 transformants (D3004), while D3207 (M230L)exhibited the same C8:0 fatty acid levels as the wild-type Cpal FATB1.D3210 (M230A), D3216 (M230T), and D3217 (M230F) all accumulated ˜1-1.5%more C8:0 than the wild-type D3004. Finally, D3132 (M2301) and D3209(M230V) exhibited a 4 fold increase in C8:0 levels compared to the D3004wild-type. While these results share some similarity with the publisheddata derived from expression in E. coli, there are some notableexceptions. For example, unlike in E. coli, substitution of M230 withLeu did not improve C8:0 fatty acid accumulation compared to thewild-type Cpal FATB1. In addition, replacing the M230 with an Ala or Thrincreased C8:0 accumulation relative to the wild-type Cpal FATB1, whichwas not expected based on the E. coli based screen.

TABLE 4 Impact on fatty acid profiles upon expression of the wild-typeCpal FATB1 or the indicated mutant within the P. moriformis algal strainS6165. Transformant C8:0 C10:0 C12:0 C14:0 Wild-type Cpal average 3.670.52 0.21 1.43 FATB1-D3004 median 2.98 0.44 0.19 1.45 M230I-D3132average 13.04 1.66 0.40 1.13 median 12.13 1.53 0.37 1.15 M230K-D3206average 0.01 0.01 0.06 1.46 median 0.00 0.00 0.06 1.45 M230L-D3207average 3.32 0.44 0.55 1.54 median 3.45 0.44 0.56 1.53 M230G-D3208average 0.05 0.01 0.07 1.58 median 0.07 0.00 0.07 1.58 M230V-D3209average 14.13 1.96 0.81 1.36 median 14.31 1.97 0.80 1.36 M230A-D3210average 4.06 0.35 0.47 1.82 median 3.92 0.34 0.45 1.80 M230R-D3211average 0.00 0.02 0.06 1.43 median 0.00 0.00 0.06 1.44 M230H-D3212average 0.00 0.05 0.10 1.49 median 0.00 0.05 0.09 1.47 M230P-D3213average 1.78 0.54 1.24 2.85 median 1.65 0.52 1.20 2.77 M230D-D3214average 0.00 0.03 0.05 1.50 median 0.00 0.03 0.05 1.50 M230E-D3215average 0.00 0.00 0.05 1.49 median 0.00 0.00 0.05 1.46 M230T-D3216average 5.83 0.57 0.39 1.48 median 5.77 0.57 0.40 1.52 M230F-D3217average 5.75 0.97 0.93 1.78 median 5.24 0.91 0.89 1.77 S6165 parent 0 00 1.50 Data shown is the average and median of 12-24 individualtransformants for each Cpal FATB1 expression construct.

Due to the discrepancies in outcome between the E. coli and P.moriformis expression, we explored the consequence of generating mutantsat the parallel position within C8-C10 specific FATB thioesterasesderived from C. hookeriana (Ch FATB2) and C. avigera (Ca FATB1). FIG. 2shows the results of replacing the Met with Iso in the Ch FATB2 (D3455,M228I) and Lys with Met or Iso in the Ca FATB1 (D3458 and D3459,respectively). Interestingly, the transformants expressing the Ch FATB2M228I (D3455) mutant exhibit ˜50% lower total C8:0-C14:0 fatty acidscompared to wild-type Ch FATB2 (D3042) expression. Transformantsexpressing the K228M Ca FATB1 (D3458) produced ˜1.5 fold greaterC8:0-C14:0 fatty acid level compared to the wild-type Ca FATB1 (D3456),while the K228I Ca FATB1 (D3459) was slightly less effective thanwild-type Ca FATB1 expression. Importantly, both K228M and K228I CaFATB1 mutants exhibited a novel fatty acid preference. Both Ca FATB1mutants accumulated a lower percent of C8:0 relative to the totalC8:0-C14:0 compared to the wild-type Ca FATB1. In addition,transformants expressing the K228I Ca FATB1 mutant (D3459) producedC12:0 and C14:0 fatty acids which was not observed with the wild-type orK228M Ca FATB1.

In summary, we have shown that the conclusions drawn from the e. coliexpression screen only partially agrees with our data derived fromexpressing the Cpal FATB1 mutants in our P. moriformis platform. Inaddition, the phenotypes observed upon substitution of the same aminoacid position within the Ch FATB2 and Ca FATB1 are not what would havebeen expected based on the original e. coli expression screen. Ourresults demonstrate that the expression platform and the thioesteraseinfluence the outcome of a mutagenesis study.

Example 4

In addition to the results shown in Table 3 we discovered thatPrototheca moriformis transformants expressing Ch FATB2 H163Y, L186P,and 230K (D3599) mutants exhibited a shift in fatty acid profilespecificity relative to the best Ch FATB2 mutant (D3599). Therefore anadditional set of mutants were generated to alter the activity andspecificity of Ch FATB2, Table 5. The 228K mutation corresponds toposition 230 of Cuphea palustris FATB1 (SEQ ID NO: 13). Residue 230 ofCuphea palustris FATB1 corresponds to M228 in the Cuphea hookerianaFATB2 and Cuphea avigera FATB1.

TABLE 5 Beneficial Mutations Constructs 1) 163Tyr 186Pro 228Lys 2)163Tyr 186Lys 228Lys 3) 163Tyr 186Ala 228Lys 4) 163Phe 186Pro 228Lys 5)163Phe 186Lys 228Lys 6) 163Phe 186Ala 228Lys

Five individual Ch FATB2 variants were generated and their effect onC8:0 and C10:0 fatty acid accumulation was quantified.

TABLE 6 Ch FABT2 variant C8:0 C10:0 C12:0 C14:0 C8-C10sum D3886-163F,average 3.72 2.33 0.20 1.80 8.05 186A, 228K STDEV 1.06 0.46 0.03 0.101.64 D3887-163F, average 5.16 2.97 0.25 1.88 10.25 186K, 228K STDEV 1.340.61 0.04 0.12 2.11 D3888-163F, average 4.57 2.72 0.18 1.85 9.32 186P,228K STDEV 1.42 0.71 0.06 0.07 2.24 D3895-163Y, average 4.17 2.51 0.201.84 8.71 186A, 228K STDEV 1.72 0.93 0.07 0.10 2.80 D3896-163Y, average4.35 2.70 0.22 1.80 9.06 186K, 228K STDEV 0.73 0.28 0.02 0.07 1.08D3598- wild type average 1.14 5.72 0.15 1.56 8.57 Ch FATB2 STDEV 0.270.77 0.06 0.05 1.12 D3519- H163Y, average 6.27 3.57 0.22 1.89 11.94L186P, 228K STDEV 2.10 0.86 0.05 0.08 3.07

Example 5

In the example below, we demonstrate the ability to modify the activityand specificity of a FATA thioesterase originally isolated from Garciniamangostana (GmFATA, accession O04792), using site directed mutagenesistargeting six amino acid positions within the enzyme. The rational fortargeting three of the amino acids (G108, S111, V193) was based onresearch published by Facciotti, et al., Nat Biotechnol. (1999)17(6):593-7. The remaining three amino acids (L91, G96, T156) weretargeted based on research performed at Solazyme with otherthioesterases.

To test the impact of each mutation on the activity of the GmFATA, thewild-type and mutant genes were cloned into a vector enabling expressionwithin the P. moriformis strain S3150. Table 7 summarizes the resultsfrom a three day lipid profile screen comparing the wild-type GmFATAwith the 14 mutants. Three GmFATA mutants (D3998, D4000, D4003)increased the amount of C18:0 by at least 1.5 fold compared to thewild-type protein (D3997). D3998 and D4003 were mutations that had beendescribed by Facciotti et al (NatBiotech 1999) as substitutions thatincreased the activity of the GmFATA. In contrast, the D4000 mutationwas based on research at Solazyme which demonstrated this positioninfluenced the activity of the FATB thioesterases.

TABLE 7 Algal SEQ ID Strain DNA # NO: GmFATA C14:0 C16:0 C18:0 C18:1C18:2 P. moriformis — — — 1.63 29.82 3.08 55.95 7.22 S3150 D3997 15Wild- 1.79 29.28 7.32 52.88 6.21 Type GmFATA D3998 16 S111A, 1.84 28.8811.19 49.08 6.21 V193A D3999 17 S111V, 1.73 29.92 3.23 56.48 6.46 V193AD4000 18 G96A 1.76 30.19 12.66 45.99 6.01 D4001 19 G96T 1.82 30.60 3.5855.50 6.28 D4002 20 G96V 1.78 29.35 3.45 56.77 6.43 D4003 21 G108A 1.7729.06 12.31 47.86 6.08 D4007 25 G108V 1.81 28.78 5.71 55.05 6.26 D400422 L91F 1.76 29.60 6.97 53.04 6.13 D4005 23 L91K 1.87 28.89 4.38 56.246.35 D4006 24 L91S 1.85 28.06 4.81 56.45 6.47 D4008 26 T156F 1.81 28.713.65 57.35 6.31 D4009 27 T156A 1.72 29.66 5.44 54.54 6.26 D4010 28 T156K1.73 29.95 3.17 56.86 6.21 D4011 29 T156V 1.80 29.17 4.97 55.44 6.27

Example 6

Wild-type P. moriformis storage lipid is comprised of ˜60% oleic(C18:1), ˜25-30% palmitic (C16:0), and ˜5-8% linoleic (C18:2) acids,with minor amounts of stearic (C18:0), myristic (C14:0), α-linolenic(C18:3α), and palmitoleic (C16:1) acids. This fatty acid profile resultsfrom the relative activities and substrate affinities of the enzymes ofthe endogeneous fatty acid biosynthetic pathway. The introduction ofGarcinia mangostana FATA thioesterase (GarmFATA1) gene into P.moriformis results in oils with increased levels of stearate (C18:0).Furthermore we demonstrated that the G96A and G108A single mutations,and the (S111A, V193A) double mutations in GarmFATA1 increased C18:0accumulation relative to the native GarmFATA1 protein.

In the present example we assessed the thioesterase activity of a seriesof additional GarmFATA1 mutants. These mutants were generated bycombining the above-described G96A, G108A, S111A and V193A mutationsinto double, triple or quadruple mutants. Specifically we testedGarmFATA1 (G96A, G108A), GarmFATA1 (G96A, S111A), GarmFATA1 (G96A,V193A), GarmFATA1 (G108A, S111A), GarmFATA1 (G108A, V193A), GarmFATA1(G96A, G108A, S111A), GarmFATA1 (G96A, G108A, V193A), GarmFATA1 (G96A,S111A, V193A), GarmFATA1 (G108A, S111A, V193A), and GarmFATA1 (G96A,G108A, S111A, V193A) mutant combinations. GarmFATA1 (G108A) was used asa control since out of all the mutants tested earlier this one gave thebest increase in C18:0 levels over the native GARMFATA1 protein. Thescreen was carried out in S5780, a strain previously constructed inS5100—a high oleic base strain. S5780 was created through the targeteddeletion of the dominant SAD2-1 allele, reducing the rate of conversionof C18:0 to C18:1 and overexpression of PmKASII, increasing elongationof C16:0 to C18:0. S5780 was transformed with constructs that targetedthe LPAT2 gene from T. cacao (TcLPAT2) and the above-mentionedcombinations of GarmFATA1 site-directed mutants to the FATA-1 locus.TcLPAT2 is highly specific for incorporation of unsaturated fatty acidsat the sn-2 position of TAGs. The S5780 strain, containing a deletion ofa stearoyl ACP desaturase (SAD) allele, was made according to theteachings in co-owned applications WO2010/063031, WO2011/150411, and/orWO2012/106560, all of which are herein incorporated by reference.

Construct Used for the Expression of TcLPAT2 and GarmFATA1 (G96A, G108A)at PmFATA1 Locus—(pSZ5990)

In this example S5780 strain, transformed with the construct pSZ5990,was generated which express Saccharomyces carlbergenesis SUC2 gene(allowing for their selection and growth on medium containing sucrose),a T. cacao LPAT2 and G. mangostana FATA1 (G96A, G108A) thioesterasetargeted at endogenous PmFATA1 genomic region. Construct pSZ5990introduced for expression in S5780 can be written as FATA-1 3′flank::CrTub2-ScSUC2-PmPGH:Spacer1:PmG3PDH-1-TcLPAT2-PmATP:Spacer2:PmSAD2-2v2-CpSAD1tp_GarmFATA1(G96A,G108A)_FLAG-PmSAD2-1::FATA-1 5′ flank

The sequence of the transforming DNA is provided in FIG. 1. Relevantrestriction sites in the construct are indicated in lowercase,underlined bold, and are from 5′-3′ BspQI, KpnI, XbaI, MfeI, BamHI,AvrII, NdeI, NsiI, AflII, EcoRI, SpeI, AscII, ClaI, SacI and BspQIrespectively. BspQI sites delimit the 5′ and 3′ ends of the transformingDNA. Bold, lowercase sequences represent genomic DNA from P. moriformisthat permit targeted integration at the FATA1 locus via homologousrecombination. Proceeding in the 5′ to 3′ direction, the Chlorellareinhardtii Tubulin 2, driving the expression of the S. cervisiae SUC2gene is indicated by lowercase, boxed text. Uppercase italics indicatethe initiator ATG and terminator TGA for SUC2, while the coding regionis indicated with lowercase italics. The P. moriformis Phosphoglyceratedehydratase (PGH) gene 3′ UTR is indicated by lowercase underlined textfollowed by buffer/spacer-1 DNA sequence indicated by lowercase bolditalic text. Immediately following the buffer nucleotide is anendogenous G3PDH-1 promoter of P. moriformis, indicated by boxedlowercase text. Uppercase italics indicate the Initiator ATG andterminator TGA codons of the T. cacao LPAT2 gene, while the lowercaseitalics indicate the remainder of the gene. The P. moriformis Adenosinetriphosphate (ATP) gene 3′ UTR is indicated by lowercase underlined textfollowed by the buffer/spacer 2 nucleotide sequence indicated bylowercase bold italic text. Immediately following the spacer-2 sequenceis the endogenous PmSAD2-2 promoter of P. moriformis, indicated by boxedlowercase text. Uppercase italics indicate the initiator ATG andterminator TGA for G. mangostana FATA1 gene while the coding region isindicated with lowercase italics. The FATA1 gene is translationallyfused to C. protothecoides Stearoyl ACP Desaturase-1 (CpSAD1) transitpeptide at the N terminal (indicated by underlined lowercase italictext) for proper targeting to chloroplast and the 3XFLAG tag at the Cterminal (indicated double underlined, italic, bold lowercase text).GarmFATA1 with CpSAD transit peptide and 3XFLAG sequence is followed byendogenous Stearoyl ACP Desaturase-1 (SAD1) gene 3′ UTR indicated bylowercase underlined text. The genomic sequence of endogenous FATA1 geneis indicated by lowercase bold text. The final construct was sequencedto ensure correct reading frames and targeting sequences, and isprovided as SEQ ID NO:46.

In addition to T. cacao LPAT2 and G. mangostana FATA1 (G96A, G108A)genes targeted at PmFAFA1 locus (pSZ5990) several other constructsincorporating the various mutations described above were designed fortransformation into S5780. These constructs are summarized below inTable 8:

TABLE 8 SEQ ID Plasmid NO: Genotype pSZ5936 47FATA-1::CrTUB2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmSAD2-2-CpSAD1_GarmFATA1(G108A)_FLAG-PmSAD2-1::FATA-1_5′ pSZ5991 48FATA-1_3′::CrTUB2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmSAD2-2-CpSAD1_GarmFATA1(G96A, S111A)_FLAG-PmSAD2-1::FATA-1_5′ pSZ5986 49FATA-1_3′::CrTUB2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmSAD2-2-CpSAD1_GarmFATA1(G96A, V193A)_FLAG-PmSAD2-1::FATA-1_5′ pSZ5982 50FATA-1_3′::CrTUB2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmSAD2-2-CpSAD1_GarmFATA1(G108A, S111A)_FLAG-PmSAD2-1::FATA-1_5′ pSZ5983 51FATA-1_3′::CrTUB2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmSAD2-2-CpSAD1_GarmFATA1(G108A, V193A)_FLAG-PmSAD2-1::FATA-1_5′ pSZ6005 52FATA-1_3′::CrTUB2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmSAD2-2-CpSAD1_GarmFATA1(G96A, G108A, S111A)_FLAG-PmSAD2-1::FATA-1_5′ pSZ5984 53FATA-1_3′::CrTUB2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmSAD2-2-CpSAD1_GarmFATA1(G96A, G108A, V193A)_FLAG-PmSAD2-1::FATA-1_5′ pSZ6004 54FATA-1_3′::CrTUB2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmSAD2-2-CpSAD1_GarmFATA1(G96A, S111A, V193A)_FLAG-PmSAD2-1::FATA-1_5′ pSZ5985 55FATA-1_3′::CrTUB2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmSAD2-2-CpSAD1_GarmFATA1(G108A, S111A, V193A)_FLAG-PmSAD2-1::FATA-1_5′ pSZ598756 FATA-1_3′::CrTUB2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmSAD2-2-CpSAD1_GarmFATA1(G96A, G108A, S111A, V193A)_FLAG-PmSAD2-1::FATA-1_5′pSZ6018 47 FATA-1::CrTUB2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmACPp1pCpSAD1_GarmFATA1(G108A)_FLAG-PmSAD2-1::FATA-1_5′ pSZ6019 50FATA-1::CrTub2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmACP-P1p-CpSAD1tp_GarmFATA1(G108A, S111A)_FLAG-PmSAD2-1:FATA-1 pSZ6020 51FATA-1::CrTub2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmACP-P1p-CpSAD1tp_GarmFATA1(G108A, V193A)_FLAG-PmSAD2-1::FATA-1 pSZ6021 53FATA-1::CrTub2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmACP-P1p-CpSAD1tp_GarmFATA1(G96A, G108A,V193A)_FLAG-PmSAD2-1::FATA-1 pSZ6022 55FATA-1::CrTub2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmACP-P1p-CpSAD1tp_GarmFATA1(G108, S111A, V193A)_FLAG-PmSAD2-1::FATA-1 pSZ6023 49FATA-1::CrTub2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmACP-P1p-CpSAD1tp_GarmFATA1(G96A, V193A)_FLAG-PmSAD2-1::FATA-1 pSZ6026 48FATA-1::CrTub2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmACP-P1p-CpSAD1tp_GarmFATA1(G96A, S111A)_FLAG-PmSAD2-1:FATA-1 pSZ6028 52FATA-1::CrTub2-ScSUC2-PmPGH:PmG3PDH-1-TcLPAT2-PmATP:PmACP-P1p-CpSAD1tp_GarmFATA1(G96A G108A, S111A) _FLAG-PmSAD2-1::FATA-1

All these constructs have the same vector backbone, selectable marker,promoters, genes and 3′ UTRs as pSZ5990 differing only in the mutationsin the GarmFATA1. In addition, the constructs pSZ6019 t0 pSZ6023,pSZ6026 and pSZ6028 differ in promoter driving the particular GarmFATA1mutant. While in pSZ5990 GarmFATA1 (G96A, G108A) is driven by PmSAD2-2v2promoter, the various GarmFATA1 mutant combinations in pSZ6019-pSZ6028are driven by PmACP-P1 promoter. The nucleotide sequences of variousGarmFATA1 mutants used in the above constructs are shown in SEQ ID NOS:47-56. The promoter sequence of PmACP-P1 is pSZ6019-pSZ6028 is shown inSEQ ID NO: 57. Relevant restriction sites as bold text are shown 5′-3′respectively.

To determine their impact on fatty acid profiles, all the constructsdescribed above were transformed independently into either S5780.Primary transformants were clonally purified and grown under standardlipid production conditions at pH5.0. The resulting profiles from a setof representative clones arising from transformation of S5780 withpSZ5936 (D4933), pSZ5990 (D4950), pSZ5991 (D4951), pSZ5986 (D4948),pSZ5982 (4931), pSZ5983 (D4932), pSZ6005 (D4952), pSZ5984 (D4933),pSZ6004 (D4953), pSZ5985 (D4934), pSZ5987 (D4949), pSZ6018 (D4978),pSZ6019 (D4979), pSZ6020 (D4980), pSZ6021 (D4981), pSZ6022 (D4982),pSZ6023 (D4983), pSZ6026 (D4986), pSZ6028 (D4988) are shown in Tables9-19 respectively.

Table 13 lists the average fatty acid profiles and glucose consumption(relative to the S7485 base strain) for each set of transformants.Disruption of one allele of FATA-1 reduces C16:0 by 1-2%, while TcLPAT2activity manifests as a 1-1.5% increase in C18:2 in these strains. D4993and D4978 expressing GarmFATA1 (G108A) mutant accumulated between 44.69%to 45.33% and 34.26 to 50.94% C18:0 respectively. D4993 has GarmFATA1(G108A) driven by PmSAD2-2 promoter while for D4978 PmACP-P1 promoterdrives the GarmFATA1 (G108A). Strains with the (G96A, G108A), (G108A,S111A) and (G108A, V193A) combinations consistently accumulated moreC18:0 than the (G108A) single mutant, with minimal increases in C16:0.D4950 (G96A, G108A) produced more than 50% C18:0 in multiple strains.The (G96A, G108A, S111A), (G96A, G108A, V193A) and (G96A, S111A, V193A)triple mutants also produced generally higher C18:0, but at a cost ofincreased C16:0. The (G108A, S111A, V193A) triple mutant and (G96A,G108A, S111A, V193A) quadruple mutant produced C18:0 less than the G108single mutant. PmACP-P1 promoter generally resulted in more C18:0 thanthe ones driven by PmSAD2-2 promoter.

TABLE 9 Fatty acid profile Sample ID C14:0 C16:0 C18:0 C18:1 C18:2C18:3α C20:0 S5780 0.75 7.07 30.32 51.61 5.96 0.79 2.16 S5780; T1402;D4993-7 0.65 4.66 45.33 38.86 7.42 0.64 1.50 S5780; T1402; D4993-4 0.664.62 45.22 38.64 7.70 0.67 1.51 S5780; T1402; D4993-2 0.63 4.54 44.9439.11 7.59 0.66 1.54 S5780; T1402; D4993-8 0.65 4.52 44.92 39.22 7.620.65 1.50 S5780; T1402; D4993-9 0.64 4.60 44.69 39.45 7.52 0.64 1.48S5780; T1395; D4978-1 0.72 5.22 50.94 32.58 7.49 0.67 1.43 S5780; T1395;D4978-6 0.68 5.15 49.26 34.74 7.17 0.65 1.45 S5780; T1395; D4978-2 0.786.21 43.12 39.62 7.01 0.72 1.57 S5780; T1395; D4978-3 0.79 6.90 34.2648.01 6.41 0.79 1.91

Table 9 provides primary 3-day Fatty acid profiles of representativeS5780 strains transformed with D4993 (pSZ5936) and D4978 (pSZ6018). BothpSZ5936 and pSZ6018 have GarmFATA1 (G108) mutant driven by PmSAD2-2 orPmACP-P1 respectively.

TABLE 10 Fatty acid profile Sample ID C14:0 C16:0 C18:0 C18:1 C18:2C18:3α C20:0 S5780 0.70 6.98 30.82 51.28 5.80 0.77 2.27 S5780; T1402;D4950-3 0.64 5.16 50.73 32.92 7.34 0.62 1.41 S5780; T1402; D4950-8 0.645.17 50.63 33.10 7.28 0.62 1.41 S5780; T1402; D4950-5 0.66 5.20 50.2333.31 7.42 0.62 1.40 S5780; T1402; D4950-7 0.65 5.15 49.90 33.81 7.310.63 1.40 S5780; T1402; D4950-4 0.66 5.22 49.53 34.13 7.21 0.61 1.42

Table 10 provides primary 3-day Fatty acid profiles of representativeS5780 strains transformed with D4950 (pSZ5990). pSZ5990 expressesGarmFATA1 (G96A, G108) mutant driven by PmSAD2-2.

TABLE 11 Fatty acid profile Sample ID C14:0 C16:0 C18:0 C18:1 C18:2C18:3 α C20:0 S5780 0.81 7.56 31.15 50.19 6.12 0.82 2.18 S5780; T1402;D4951-18 0.73 5.11 46.22 36.56 7.92 0.72 1.54 S5780; T1402; D4951-8 0.704.80 42.65 40.59 7.77 0.72 1.58 S5780; T1402; D4951-3 0.70 4.82 42.4240.74 7.76 0.71 1.58 S5780; T1402; D4951-4 0.69 4.82 42.28 40.88 7.760.73 1.60 S5780; T1402; D4951-15 0.72 4.95 42.07 40.72 8.00 0.73 1.58S5780; T1395; D4986-21 0.79 5.78 48.77 33.99 7.54 0.69 1.48 S5780;T1395; D4986-18 0.77 5.77 48.43 34.61 7.32 0.65 1.46 S5780; T1395;D4986-23 0.78 5.66 47.64 35.30 7.44 0.69 1.49 S5780; T1395; D4986-150.75 5.52 47.60 35.80 7.21 0.67 1.50 S5780; T1395; D4986-1 0.84 6.3846.95 34.55 8.29 0.64 1.33

Table 11 provides Primary 3-day Fatty acid profiles of representativeS5780 strains transformed with D4951 (pSZ5991) and D4986 (pSZ6026).pSZ5991 and pSZ6026 express GarmFATA1 (G96A, S111A) mutant driven byPmSAD2-2 and PmACP-P1 respectively.

TABLE 12 Fatty acid profile Sample ID C14:0 C16:0 C18:0 C18:1 C18:2C18:3α C20:0 S5780 0.82 7.51 30.86 50.34 6.27 0.86 2.16 S5780; T1388;D4948-6 0.70 4.93 46.65 36.92 7.66 0.68 1.50 S5780; T1388; D4948-10 0.664.79 46.23 37.72 7.51 0.68 1.43 S5780; T1388; D4948-11 0.72 5.05 46.1836.89 8.04 0.67 1.47 S5780; T1388; D4948-4 0.72 5.11 46.11 36.97 8.000.66 1.45 S5780; T1388; D4948-9 0.72 5.06 46.09 36.96 8.05 0.67 1.45S5780; T1395; D4983-25 0.73 5.85 49.47 32.96 7.77 0.62 1.49 S5780;T1395; D4983-14 0.68 5.25 48.53 35.02 7.32 0.63 1.52 S5780; T1395;D4983-27 0.70 5.66 48.35 34.56 7.56 0.62 1.50 S5780; T1395; D4983-180.67 5.30 48.26 35.35 7.29 0.62 1.51 S5780; T1395; D4983-13 0.68 5.3148.09 35.59 7.27 0.63 1.48

Table 12 provides primary 3-day Fatty acid profiles of representativeS5780 strains transformed with D4948 (pSZ5986) and D4983 (pSZ6023).pSZ5986 and pSZ6023 express GarmFATA1 (G96A, V193A) mutant driven byPmSAD2-2 and PmACP-P1 respectively.

TABLE 13 Fatty acid profile Sample ID C14:0 C16:0 C18:0 C18:1 C18:2C18:3α C20:0 S5780 0.77 7.36 30.84 50.71 6.24 0.87 2.12 S5780; T1388;D4931-25 0.70 5.13 48.48 35.40 7.25 0.64 1.43 S5780; T1388; D4931-9 0.735.43 48.29 34.92 7.63 0.65 1.41 S5780; T1388; D4931-13 0.72 5.24 48.1335.22 7.64 0.66 1.45 S5780; T1388; D4931-17 0.76 5.14 48.07 35.08 7.860.68 1.42 S5780; T1388; D4931-12 0.73 5.33 47.91 35.27 7.65 0.67 1.42S5780; T1395; D4979-36 0.89 6.91 50.03 31.13 7.83 0.67 1.40 S5780;T1395; D4979-5 0.77 5.88 49.65 33.24 7.25 0.68 1.48 S5780; T1395;D4979-41 0.79 6.25 49.52 33.09 7.28 0.63 1.42 S5780; T1395; D4979-390.82 6.36 49.43 32.49 7.66 0.66 1.48 S5780; T1395; D4979-32 0.82 6.4949.12 32.98 7.45 0.63 1.43

Table 13 provides primary 3-day Fatty acid profiles of representativeS5780 strains transformed with D4931 (pSZ5982) and D4979 (pSZ6019).pSZ5982 and pSZ6019 express GarmFATA1 (G108A, S111A) mutant driven byPmSAD2-2 and PmACP-P1 respectively.

TABLE 14 Fatty acid profile Sample ID C14:0 C16:0 C18:0 C18:1 C18:2C18:3α C20:0 S5780 0.79 7.46 31.60 49.57 6.28 0.86 2.19 S5780; T1388;D4932-48 0.78 4.59 49.48 31.74 9.13 1.30 1.84 S5780; T1388; D4932-360.66 4.89 49.25 34.63 7.53 0.66 1.43 S5780; T1388; D4932-28 0.66 4.9349.04 34.91 7.50 0.65 1.38 S5780; T1388; D4932-23 0.67 4.95 49.03 34.557.69 0.66 1.42 S5780; T1388; D4932-5 0.68 4.93 49.01 34.77 7.54 0.671.38 S5780; T1395; D4980-21 0.71 4.54 51.48 32.09 7.54 0.87 1.78 S5780;T1395; D4980-1 0.72 5.80 48.65 33.81 8.04 0.62 1.46 S5780; T1395;D4980-25 0.68 5.46 47.67 35.53 7.61 0.66 1.47 S5780; T1395; D4980-180.77 6.49 46.51 34.39 8.69 0.71 1.45 S5780; T1395; D4980-30 0.70 5.2245.14 38.84 6.80 0.70 1.70

Table 14 provides primary 3-day Fatty acid profiles of representativeS5780 strains transformed with D4932 (pSZ5983) and D4980 (pSZ6020).pSZ5983 and pSZ6020 express GarmFATA1 (G108A, V193A) mutant driven byPmSAD2-2 and PmACP-P1 respectively.

TABLE 15 Fatty acid profile Sample ID C14:0 C16:0 C18:0 C18:1 C18:2C18:3α C20:0 S5780 0.75 7.14 30.84 51.19 5.87 0.79 2.26 S5780; T1402;D4952-9 0.77 5.68 48.96 33.44 7.70 0.68 1.50 S5780; T1402; D4952-5 0.755.58 48.60 33.94 7.60 0.70 1.52 S5780; T1402; D4952-1 0.75 5.62 48.5933.94 7.63 0.69 1.51 S5780; T1402; D4952-8 0.78 5.78 48.51 33.71 7.740.67 1.50 S5780; T1402; D4952-10 0.77 5.65 48.35 34.15 7.59 0.70 1.52S5780; T1395; D4988-5 0.99 8.68 48.51 31.29 7.08 0.64 1.51 S5780; T1395;D4988-7 0.75 5.50 46.63 36.68 7.41 0.69 1.43 S5780; T1395; D4988-8 0.775.57 46.51 36.73 7.47 0.70 1.42 S5780; T1395; D4988-3 1.12 9.63 44.0633.16 8.33 0.76 1.57 S5780; T1395; D4988-10 1.27 11.45 43.35 31.26 8.950.74 1.49

Table 15 provides primary 3-day Fatty acid profiles of representativeS5780 strains transformed with D4952 (pSZ6005) and D4988 (pSZ6028).pSZ6005 and pSZ6028 express GarmFATA1 (G96A, G108A, S111A) mutant drivenby PmSAD2-2 and PmACP-P1 respectively.

TABLE 16 Fatty acid profile Sample ID C14:0 C16:0 C18:0 C18:1 C18:2C18:3α C20:0 S5780 0.79 7.46 31.60 49.57 6.28 0.86 2.19 S5780; T1388;D4933-12 0.67 5.48 50.40 32.85 7.31 0.63 1.36 S5780; T1388; D4933-9 0.695.68 50.20 32.58 7.55 0.65 1.41 S5780; T1388; D4933-8 0.66 5.46 50.0733.20 7.35 0.63 1.39 S5780; T1388; D4933-2 0.70 5.66 49.99 32.81 7.610.63 1.38 S5780; T1388; D4933-5 0.85 5.84 41.97 39.70 7.94 0.97 1.36S5780; T1395; D4981-1 0.63 5.07 37.33 46.45 6.75 0.76 2.00 S5780; T1395;D4981-3 0.71 5.70 34.96 47.88 7.02 0.86 1.88 S5780; T1395; D4981-7 0.705.87 34.44 48.58 6.52 0.78 2.04 S5780; T1395; D4981-4 0.75 6.18 33.7848.83 6.61 0.83 1.98 S5780; T1395; D4981-8 0.71 6.42 33.38 49.33 6.050.78 2.21

Table 16 provides primary 3-day Fatty acid profiles of representativeS5780 strains transformed with D4933 (pSZ5984) and D4981 (pSZ6021).pSZ5984 and pSZ6021 express GarmFATA1 (G96A, G108A, V193A) mutant drivenby PmSAD2-2 and PmACP-P1 respectively.

TABLE 17 Fatty acid profile Sample ID C14:0 C16:0 C18:0 C18:1 C18:2C18:3α C20:0 S5780 0.74 7.27 31.04 50.75 5.96 0.81 2.18 S5780; T1388;D4953-6 0.84 6.99 47.90 33.26 7.58 0.65 1.46 S5780; T1388; D4953-4 0.857.09 47.54 33.64 7.46 0.66 1.42 S5780; T1402; D4953-3 0.89 6.91 47.5433.36 7.56 0.71 1.60 S5780; T1402; D4953-9 0.91 7.26 46.67 33.52 7.900.70 1.49 S5780; T1402; D4953-1 0.90 7.20 46.37 33.86 7.91 0.72 1.54

Table 17 provides primary 3-day Fatty acid profiles of representativeS5780 strains transformed with D4953 (pSZ6004). pSZ6004 expressesGarmFATA1 (G96A, S111A, V193A) mutant driven by PmSAD2-2.

TABLE 18 Fatty acid profile Sample ID C14:0 C16:0 C18:0 C18:1 C18:2C18:3α C20:0 S5780 0.78 8.24 30.24 50.34 6.00 0.79 2.23 S5780; T1402;D4934-20 0.84 7.10 46.71 34.60 7.34 0.65 1.47 S5780; T1402; D4934-150.78 6.76 44.01 38.09 6.88 0.65 1.59 S5780; T1402; D4934-24 1.03 10.6939.82 33.95 11.12 0.71 1.36 S5780; T1402; D4934-14 0.77 6.83 38.68 43.316.51 0.71 1.88 S5780; T1402; D4934-16 0.75 6.91 35.57 46.50 6.20 0.711.92 S5780; T1395; D4982-1 0.00 6.19 39.51 41.35 8.23 0.78 1.92 S5780;T1395; D4982-2 0.03 7.02 35.52 46.24 6.59 0.81 1.89

Table 18 provides primary 3-day Fatty acid profiles of representativeS5780 strains transformed with D4934 (pSZ5985) and D4982 (pSZ6022).pSZ5985 and pSZ6022 express GarmFATA1 (G108A, S111A, V193A) mutantdriven by PmSAD2-2 and PmACP-P1 respectively.

TABLE 19 Fatty acid profile Sample ID C14:0 C16:0 C18:0 C18:1 C18:2C18:3 α C20:0 S5780 0.70 6.98 30.82 51.28 5.80 0.77 2.27 S5780; T1402;D4949-2 0.62 4.54 46.07 38.20 7.44 0.63 1.46 S5780; T1402; D4949-13 0.664.57 45.33 38.42 7.85 0.68 1.50 S5780; T1402; D4949-7 0.64 4.61 45.0239.06 7.55 0.64 1.50 S5780; T1402; D4949-8 0.64 4.62 44.87 39.16 7.510.67 1.54 S5780; T1402; D4949-3 0.64 4.88 44.18 39.83 7.18 0.65 1.56

Table 19 provides primary 3-day Fatty acid profiles of representativeS5780 strains transformed with D4949 (pSZ5987). pSZ5985 expressesGarmFATA1 (G96A, G108A, S111A, V193A) mutant driven by PmSAD2-2.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

INFORMAL SEQUENCE LISTING SEQ ID No. 1Wildtype Cuphea hookeriana FATB2 (“ChFATB2”) amino acid sequenceMVAAAASSAFFPVPAPGASPKPGKFGNWPSSLSPSFKPKSIPNGGFQVKANDSAHPKANGSAVSLKSGSLNTQEDTSSSPPPRTFLHQLPDWSRLLTAITTVFVKSKRPDMHDRKSKRPDMLVDSFGLESTVQDGLVFRQSFSIRSYEIGTDRTASIETLMNHLQETSLNHCKSTGILLDGFGRTLEMCKRDLIWVVIKMQIKVNRYPAWGDTVEINTRFSRLGKIGMGRDWLISDCNTGEILVRATSAYAMMNQKTRRLSKLPYEVHQEIVPLFVDSPVIEDSDLKVHKFKVKTGDSIQKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRRECGRDSVLESVTAMDPSKVGVRSQYQHLLRLEDGTAIVNGATEWRPKNAGANGAISTGKTSNGNSVSSEQ ID NO: 2 Cuphea hookeriana C8/C10 specific FATB2 CDSCTGGATACCATTTTCCCTGCGAAAAAACATGGTGGCTGCTGCAGCAAGTTCCGCATTCTTCCCTGTTCCAGCCCCGGGAGCCTCCCCTAAACCCGGGAAGTTCGGAAATTGGCCCTCGAGCTTGAGCCCTTCCTTCAAGCCCAAGTCAATCCCCAATGGCGGATTTCAGGTTAAGGCAAATGACAGCGCCCATCCAAAGGCTAACGGTTCTGCAGTTAGTCTAAAGTCTGGCAGCCTCAACACTCAGGAGGACACTTCGTCGTCCCCTCCTCCTCGGACTTTCCTTCACCAGTTGCCTGATTGGAGTAGGCTTCTGACTGCAATCACGACCGTGTTCGTGAAATCTAAGAGGCCTGACATGCATGATCGGAAATCCAAGAGGCCTGACATGCTGGTGGACTCGTTTGGGTTGGAGAGTACTGTTCAGGATGGGCTCGTGTTCCGACAGAGTTTTTCGATTAGGTCTTATGAAATAGGCACTGATCGAACGGCCTCTATAGAGACACTTATGAACCACTTGCAGGAAACATCTCTCAATCATTGTAAGAGTACCGGTATTCTCCTTGACGGCTTCGGTCGTACTCTTGAGATGTGTAAAAGGGACCTCATTTGGGTGGTAATAAAAATGCAGATCAAGGTGAATCGCTATCCAGCTTGGGGCGATACTGTCGAGATCAATACCCGGTTCTCCCGGTTGGGGAAAATCGGTATGGGTCGCGATTGGCTAATAAGTGATTGCAACACAGGAGAAATTCTTGTAAGAGCTACGAGCGCGTATGCCATGATGAATCAAAAGACGAGAAGACTCTCAAAACTTCCATACGAGGTTCACCAGGAGATAGTGCCTCTTTTTGTCGACTCTCCTGTCATTGAAGACAGTGATCTGAAAGTGCATAAGTTTAAAGTGAAGACTGGTGATTCCATTCAAAAGGGTCTAACTCCGGGGTGGAATGACTTGGATGTCAATCAGCACGTAAGCAACGTGAAGTACATTGGGTGGATTCTCGAGAGTATGCCAACAGAAGTTTTGGAGACCCAGGAGCTATGCTCTCTCGCCCTTGAATATAGGCGGGAATGCGGAAGGGACAGTGTGCTGGAGTCCGTGACCGCTATGGATCCCTCAAAAGTTGGAGTCCGTTCTCAGTACCAGCACCTTCTGCGGCTTGAGGATGGGACTGCTATCGTGAACGGTGCAACTGAGTGGCGGCCGAAGAATGCAGGAGCTAACGGGGCGATATCAACGGGAAAGACTTCAAATGGAAACTCGGTCTCTTAGAAGTGTCTCGGAACCCTTCCGAGATGTGCATTTCTTTTCTCCTTTTCATTTTGTGGTGAGCTGAAAGAAGAGCATGTCGTTGCAATCAGTAAATTGTGTAGTTCGTTTTTCGCTTTGCTTCGCTCCTTTGTATAATAATATGGTCAGTCGTCTTTGTATCATTTCATGTTTTCAGTTTATTTACGCCATATAATTTTT SEQ ID NO: 3Amino acid sequence of Ch FATB2-D3570, pSZ4689-the algal transitpeptide is underlined, the FLAG epitope tag is bolded and the P186residue is lower-case boldMATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRASSLSPSFKPKSIPNGGFQVKANDSAHPKANGSAVSLKSGSLNTQEDTSSSPPPRTFLHQLPDWSRLLTAITTVFVKSKRPDMHDRKSKRPDMLVDSFGLESTVQDGLVFRQSFSIRSYEIGTDRTASIETLMNHLQETSLNHCKSTGILLDGFGRTpEMCKRDLIWVVIKMQIKVNRYPAWGDTVEINTRFSRLGKIGMGRDWLISDCNTGEILVRATSAYAMMNQKTRRLSKLPYEVHQEIVPLFVDSPVIEDSDLKVHKFKVKTGDSIQKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRRECGRDSVLESVTAMDPSKVGVRSQYQHLLRLEDGTAIVNGATEWRPKNAGANGAISTGKTSNGNSVSMDYKDHDGDYKDHDIDYKDDDDK* SEQ ID NO: 4Amino acid sequence of Ch FATB2-D3573, pSZ4692-the algal transitpeptide is underlined, the FLAG epitope tag is bolded and the K186residue is lower-case boldMATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRASSLSPSFKPKSIPNGGFQVKANDSAHPKANGSAVSLKSGSLNTQEDTSSSPPPRTFLHQLPDWSRLLTAITTVFVKSKRPDMHDRKSKRPDMLVDSFGLESTVQDGLVFRQSFSIRSYEIGTDRTASIETLMNHLQETSLNHCKSTGILLDGFGRTkEMCKRDLIWVVIKMQIKVNRYPAWGDTVEINTRFSRLGKIGMGRDWLISDCNTGEILVRATSAYAMMNQKTRRLSKLPYEVHQEIVPLFVDSPVIEDSDLKVHKFKVKTGDSIQKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRRECGRDSVLESVTAMDPSKVGVRSQYQHLLRLEDGTAIVNGATEWRPKNAGANGAISTGKTSNGNSVSMDYKDHDGDYKDHDIDYKDDDDK* SEQ ID NO: 5Amino acid sequence of Ch FATB2-D3582, pSZ4702-the algal transitpeptide is underlined, the FLAG epitope tag is bolded and the A186residue is lower-case boldMATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRASSLSPSFKPKSIPNGGFQVKANDSAHPKANGSAVSLKSGSLNTQEDTSSSPPPRTFLHQLPDWSRLLTAITTVFVKSKRPDMHDRKSKRPDMLVDSFGLESTVQDGLVFRQSFSIRSYEIGTDRTASIETLMNHLQETSLNHCKSTGILLDGFGRTaEMCKRDLIWVVIKMQIKVNRYPAWGDTVEINTRFSRLGKIGMGRDWLISDCNTGEILVRATSAYAMMNQKTRRLSKLPYEVHQEIVPLFVDSPVIEDSDLKVHKFKVKTGDSIQKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRRECGRDSVLESVTAMDPSKVGVRSQYQHLLRLEDGTAIVNGATEWRPKNAGANGAISTGKTSNGNSVSMDYKDHDGDYKDHDIDYKDDDDK* SEQ ID NO: 6Amino acid sequence of Ch FATB2-D3584, pSZ4704-the algal transitpeptide is underlined, the FLAG epitope tag is bolded and the Y163residue is lower-case boldMATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRASSLSPSFKPKSIPNGGFQVKANDSAHPKANGSAVSLKSGSLNTQEDTSSSPPPRTFLHQLPDWSRLLTAITTVFVKSKRPDMHDRKSKRPDMLVDSFGLESTVQDGLVFRQSFSIRSYEIGTDRTASIETLMNyLQETSLNHCKSTGILLDGFGRTLEMCKRDLIWVVIKMQIKVNRYPAWGDTVEINTRFSRLGKIGMGRDWLISDCNTGEILVRATSAYAMMNQKTRRLSKLPYEVHQEIVPLFVDSPVIEDSDLKVHKFKVKTGDSIQKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRRECGRDSVLESVTAMDPSKVGVRSQYQHLLRLEDGTAIVNGATEWRPKNAGANGAISTGKTSNGNSVSMDYKDHDGDYKDHDIDYKDDDDK* SEQ ID NO: 7Amino acid sequence of Ch FATB2-D3588, pSZ4709-the algal transitpeptide is underlined, the FLAG epitope tag is bolded and the F163residue is lower-case boldMATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRASSLSPSFKPKSIPNGGFQVKANDSAHPKANGSAVSLKSGSLNTQEDTSSSPPPRTFLHQLPDWSRLLTAITTVFVKSKRPDMHDRKSKRPDMLVDSFGLESTVQDGLVFRQSFSIRSYEIGTDRTASIETLMNfLQETSLNHCKSTGILLDGFGRTLEMCKRDLIWVVIKMQIKVNRYPAWGDTVEINTRFSRLGKIGMGRDWLISDCNTGEILVRATSAYAMMNQKTRRLSKLPYEVHQEIVPLFVDSPVIEDSDLKVHKFKVKTGDSIQKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRRECGRDSVLESVTAMDPSKVGVRSQYQHLLRLEDGTAIVNGATEWRPKNAGANGAISTGKTSNGNSVSMDYKDHDGDYKDHDIDYKDDDDK* SEQ ID NO: 8Amino acid sequence of the wild-type Ch FATB2-D3598, pSZ4243-thealgal transit peptide is underlined, the FLAG epitope tag is boldedand the H163 and L186 residues are lower-case boldMATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRASSLSPSFKPKSIPNGGFQVKANDSAHPKANGSAVSLKSGSLNTQEDTSSSPPPRTFLHQLPDWSRLLTAITTVFVKSKRPDMHDRKSKRPDMLVDSFGLESTVQDGLVFRQSFSIRSYEIGTDRTASIETLMNhLQETSLNHCKSTGILLDGFGRTlEMCKRDLIWVVIKMQIKVNRYPAWGDTVEINTRFSRLGKIGMGRDWLISDCNTGEILVRATSAYAMMNQKTRRLSKLPYEVHQEIVPLFVDSPVIEDSDLKVHKFKVKTGDSIQKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRRECGRDSVLESVTAMDPSKVGVRSQYQHLLRLEDGTAIVNGATEWRPKNAGANGAISTGKTSNGNSVSMDYKDHDGDYKDHDIDYKDDDDK* SEQ ID NO: 9Nucleotide sequence of the ChFATB2 expression screening vector,using pSZ4243 as an example. The 5' and 3' homology arms enablingtargeted integration into the Thi4 locus are noted with lowercase;the CrTUB2 promoter is noted in uppercase italic which drivesexpression of the Neomycin resistance gene noted with lowercaseitalic followed by the PmPGH 3'UTR terminator highlighted inuppercase. The PmUAPA1 promoter (noted in bold text) drives theexpression of the codon optimized ChFATB2 (noted with lowercasebold text) and is terminated with the CvNR 3'UTR noted inunderlined, lower case bold. Restriction cloning sites and spacerDNA fragments are noted as underlined, uppercase plain lettering.ccctcaactgcgacgctgggaaccttctccgggcaggcgatgtgcgtgggtttgcctccttggcacggctctacaccgtcgagtacgccatgaggcggtgatggctgtgtcggttgccacttcgtccagagacggcaagtcgtccatcctctgcgtgtgtggcgcgacgctgcagcagtccctctgcagcagatgagcgtgactttggccatttcacgcactcgagtgtacacaatccatttttcttaaagcaaatgactgctgattgaccagatactgtaacgctgatttcgctccagatcgcacagatagcgaccatgttgctgcgtctgaaaatctggattccgaattcgaccctggcgctccatccatgcaacagatggcgacacttgttacaattcctgtcacccatcggcatggagcaggtccacttagattcccgatcacccacgcacatctcgctaatagtcattcgttcgtgtcttcgatcaatctcaagtgagtgtgcatggatcttggttgacgatgcggtatgggtttgcgccgctggctgcagggtctgcccaaggcaagctaacccagctcctctccccgacaatactctcgcaggcaaagccggtcacttgccttccagattgccaataaactcaattatggcctctgtcatgccatccatgggtctgatgaatggtcacgctcgtgtcctgaccgttccccagcctctggcgtcccctgccccgcccaccagcccacgccgcgcggcagtcgctgccaaggctgtctcggaGGTACC CTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAAC TCTAGAATATCAatgatcgagcaggacggcctccacgccggctcccccgccgcctgggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttctga CAATTGACGCCCGCGCGGCGCACCTGACCTGTTCTCTCGAGGGCGCCTGTTCTGCCTTGCGAAACAAGCCCCTGGAGCATGCGTGCATGATCGTCTCTGGCGCCCCGCCGCGCGGTTTGTCGCCCTCGCGGGCGCCGCGGCCGCGGGGGCGCATTGAAATTGTTGCAAACCCCACCTGACAGATTGAGGGCCCAGGCAGGAAGGCGTTGAGATGGAGGTACAGGAGTCAAGTAACTGAAAGTTTTTATGATAACTAACAACAAAGGGTCGTTTCTGGCCAGCGAATGACAAGAACAAGATTCCACATTTCCGTGTAGAGGCTTGCCATCGAATGTGAGCGGGCGGGCCGCGGACCCGACAAAACCCTTACGACGTGGTAAGAAAAACGTGGCGGGCACTGTCCCTGTAGCCTGAAGACCAGCAGGAGACGATCGGAAGCATCACAGCACAGGATCCCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTCGAAACGTTCACAGCCTAGGGATATC ATAGCGACTGCTACCCCCCGACCATGTGCCGAGGCAGAAATTATATACAAGAAGCAGATCGCAATTAGGCACATCGCTTTGCATTATCCACACACTATTCATCGCTGCTGCGGCAAGGCTGCAGAGTGTATTTTTGTGGCCCAGGAGCTGAGTCCGAAGTCGACGCGACGAGCGGCGCAGGATCCGACCCCTAGACGAGCACTGTCATTTTCCAAGCACGCAGCTAAATGCGCTGAGACCGGGTCTAAATCATCCGAAAAGTGTCAAAATGGCCGATTGGGTTCGCCTAGGACAATGCGCTGCGGATTCGCTCGAGTCCGCTGCCGGCCAAAAGGCGGTGGTACAGGAAGGCGCACGGGGCCAACCCTGCGAAGCCGGGGGCCCGAACGCCGACCGCCGGCCTTCGATCTCGGGTGTCCCCCTCGTCAATTTCCTCTCTCGGGTGCAGCCACGAAAGTCGTGACGCAGGTCACGAAATCCGGTTACGAAAAACGCAGGTCTTCGCAAAAACGTGAGGGTTTCGCGTCTCGCCCTAGCTATTCGTATCGCCGGGTCAGACCCACGTGCAGAAAAGCCCTTGAATAACCCGGGACCGTGGTTACCGCGCCGCCTGCACCAGGGGGCTTATATAAGCCCACACCACACCTGTCTCACCACGCATTTCTCCAACTCGCGACTTTTCGGAAGAAATTGTTATCCACCTAGTATAGACTGCCACCTGCAGGACCTTGTGTCTTGCAGTTTGTATTGGTCCCGGCCGTCGAGCACGACAGATCTGGGCTAGGGTTGGCCTGGCCGCTCGGCACTCCCCTTTAGCCGCGCGCATCCGCGTTCCAGAGGTGCGATTCGGTGTGTGGAGCATTGTCATGCGCTTGTGGGGGTCGTTCCGTGCGCGGCGGGTCCGCCATGGGCGCCGACCTGGGCCCTAGGGTTTGTTTTCGGGCCAAGCGAGCCCCTCTCACCTCGTCGCCCCCCCGCATTCCCTCTCTCTTGCAGCC ACTAGTAACA atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgcctccagcctgagcccctccttcaagcccaagtccatccccaacggcggcttccaggtgaaggccaacgacagcgcccaccccaaggccaacggctccgccgtgagcctgaagagcggcagcctgaacacccaggaggacacctcctccagcccccccccccgcaccttcctgcaccagctgcccgactggagccgcctgctgaccgccatcaccaccgtgttcgtgaagtccaagcgccccgacatgcacgaccgcaagtccaagcgccccgacatgctggtggacagcttcggcctggagtccaccgtgcaggacggcctggtgttccgccagtccttctccatccgctcctacgagatcggcaccgaccgcaccgccagcatcgagaccctgatgaaccacctgcaggagacctccctgaaccactgcaagagcaccggcatcctgctggacggcttcggccgcaccctggagatgtgcaagcgcgacctgatctgggtggtgatcaagatgcagatcaaggtgaaccgctaccccgcctggggcgacaccgtggagatcaacacccgcttcagccgcctgggcaagatcggcatgggccgcgactggctgatctccgactgcaacaccggcgagatcctggtgcgcgccaccagcgcctacgccatgatgaaccagaagacccgccgcctgtccaagctgccctacgaggtgcaccaggagatcgtgcccctgttcgtggacagccccgtgatcgaggactccgacctgaaggtgcacaagttcaaggtgaagaccggcgacagcatccagaagggcctgacccccggctggaacgacctggacgtgaaccagcacgtgtccaacgtgaagtacatcggctggatcctggagagcatgcccaccgaggtgctggagacccaggagctgtgctccctggccctggagtaccgccgcgagtgcggccgcgactccgtgctggagagcgtgaccgccatggaccccagcaaggtgggcgtgcgctcccagtaccagcacctgctgcgcctggaggacggcaccgccatcgtgaacggcgccaccgagtggcgccccaagaacgccggcgccaacggcgccatctccaccggcaagaccagcaacggcaactccgtgtccatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga CTCGAGgcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatttacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaAAGCTGTATAGGGATAACAGGGTAATGAGCTCcagcgccatgccacgccctttgatggcttcaagtacgattacggtgttggattgtgtgtttgttgcgtagtgtgcatggtttagaataatacacttgatttcttgctcacggcaatctcggcttgtccgcaggttcaaccccatttcggagtctcaggtcagccgcgcaatgaccagccgctacttcaaggacttgcacgacaacgccgaggtgagctatgtttaggacttgattggaaattgtcgtcgacgcatattcgcgctccgcgacagcacccaagcaaaatgtcaagtgcgttccgatttgcgtccgcaggtcgatgttgtgatcgtcggcgccggatccgccggtctgtcctgcgcttacgagctgaccaagcaccctgacgtccgggtacgcgagctgagattcgattagacataaattgaagattaaacccgtagaaaaatttgatggtcgcgaaactgtgctcgattgcaagaaattgatcgtcctccactccgcaggtcgccatcatcgagcagggcgttgctcccggcggcggcgcctggctggggggacagctgttctcggccatgtgtgtacgtagaaggatgaatttcagctggttttcgttgcacagctgtttgtgcatgatttgtttcagactattgttgaatgtttttagatttcttaggatgcatgatttgtctgcatgcgact SEQ ID NO: 10Amino acid sequence of the wild-type Cpal FATB1 (D3004, pSZ4241).The algal transit peptide is underlined, the FLAG epitope tag isbolded and the M230 residue is lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRASSSLSPSLKPKSIPNGGFQVKANASAHPKANGSAVTLKSGSLNTQEDTLSSSPPPRAFFNQLPDWSMLLTAITTVFVAPEKRWTMFDRKSKRPNMLMDSFGLERVVQDGLVFRQSFSIRSYEICADRTASIETVMNHVQETSLNQCKSIGLLDDGFGRSPEMCKRDLIWVVTRMKIMVNRYPTWGDTIEVSTWLSQSGKIGmGRDWLISDCNTGEILVRATSVYAMMNQKTRRFSKLPHEVRQEFAPHFLDSPPAIEDNDGKLQKFDVKTGDSIRKGLTPGWYDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLTLEYRRECGRDSVLESVTSMDPSKVGDRFQYRHLLRLEDGADIMKGRTEWRPKNAGTNGAISTGKTMDYKDHDGDYKDHDIDYKDDDDK SEQ ID NO: 11Amino acid sequence of the wild-type Chook FATB2 (D3042, pSZ4243).The algal transit peptide is underlined, the FLAG epitope tag isbolded and the M228 residue is lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRASSLSPSFKPKSIPNGGFQVKANDSAHPKANGSAVSLKSGSLNTQEDTSSSPPPRTFLHQLPDWSRLLTAITTVFVKSKRPDMHDRKSKRPDMLVDSFGLESTVQDGLVFRQSFSIRSYEIGTDRTASIETLMNHLQETSLNHCKSTGILLDGFGRTLEMCKRDLIWVVIKMQIKVNRYPAWGDTVEINTRFSRLGKIGmGRDWLISDCNTGEILVRATSAYAMMNQKTRRLSKLPYEVHQEIVPLFVDSPVIEDSDLKVHKFKVKTGDSIQKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRRECGRDSVLESVTAMDPSKVGVRSQYQHLLRLEDGTAIVNGATEWRPKNAGANGAISTGKTSNGNSVSMDYKDHDGDYKDHDIDYKDDDDK SEQ ID NO: 12Amino acid sequence of the wild-type Ca FATB1 (D3456, pSZ4532). Thealgal transit peptide is underlined, the FLAG epitope tag is boldedand the K228 residue is lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRAAINSRAHPKANGSAVSLKSGSLNTQEDTSSSPPPRTFLHQLPDWSRLLTAITTVFVKSKRPDMHDRKSKRPDMLMDSFGLESIVQEGLEFRQSFSIRSYEIGTDRTASIETLMNYLQETSLNHCKSTGILLDGFGRTPEMCKRDLIWVVTKMKIKVNRYPAWGDTVEINTWFSRLGKIGkGRDWLISDCNTGEILIRATSAYATMNQKTRRLSKLPYEVHQEIAPLFVDSPPVIEDNDLKLHKFEVKTGDSIHKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRRECGRDSVLESVTAMDPTKVGGRSQYQHLLRLEDGTDIVKCRTEWRPKNPGANGAISTGKTSNGNSVSMDYKDHDGDYKDHDIDYKDDDDK SEQ ID NO: 13Amino acid sequence of Cuphea palustris FATB1MVAAAASSACFPVPSPGASPKPGKLGNWSSSLSPSLKPKSIPNGGFQVKANASAHPKANGSAVTLKSGSLNTQEDTLSSSPPPRAFFNQLPDWSMLLTAITTVFVAPEKRWTMFDRKSKRPNMLMDSFGLERVVQDGLVFRQSFSIRSYEICADRTASIETVMNHVQETSLNQCKSIGLLDDGFGRSPEMCKRDLIWVVTRMKIMVNRYPTWGDTIEVSTWLSQSGKIGMGRDWLISDCNTGEILVRATSVYAMMNQKTRRFSKLPHEVRQEFAPHFLDSPPAIEDNDGKLQKFDVKTGDSIRKGLTPGWYDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLTLEYRRECGRDSVLESVTSMDPSKVGDRFQYRHLLRLEDGADIMKGRTEWRPKNAGTNGAISTGKT SEQ ID NO: 14Amino acid sequence of Cuphea avigera FATB1MVAAAASSAFFSVPVPGTSPKPGKFRIWPSSLSPSFKPKPIPNGGLQVKANSRAHPKANGSAVSLKSGSLNTQEDTSSSPPPRTFLHQLPDWSRLLTAITTVFVKSKRPDMHDRKSKRPDMLMDSFGLESIVQEGLEFRQSFSIRSYEIGTDRTASIETLMNYLQETSLNHCKSTGILLDGFGRTPEMCKRDLIWVVTKMKIKVNRYPAWGDTVEINTWFSRLGKIGKGRDWLISDCNTGEILIRATSAYATMNQKTRRLSKLPYEVHQEIAPLFVDSPPVIEDNDLKLHKFEVKTGDSIHKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRRECGRDSVLESVTAMDPTKVGGRSQYQHLLRLEDGTDIVKCRTEWRPKNPGANGAISTGKTSNGNSVS SEQ ID NO: 15Amino acid sequence of Gm FATA wild-type parental gene; D3997,pSZ5083. The algal transit peptide is underlined and the FLAGepitope tag is uppercase boldMATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYKYPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHDGDYKDHDIDYKDDDDK SEQ ID NO: 16Amino acid sequence of Gm FATA S111A, V193A mutant gene; D3998,pSZ5084. The algal transit peptide is underlined, the FLAG epitopetag is uppercase bold and the S111A, V193A residues are lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFaTTPTMRKLRLIWVTARMHIEIYKYPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDaDVRDEYLVHCPRELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHDGDYKDHDIDYKDDDDK SEQ ID NO: 17Amino acid sequence of Gm FATA S111V, V193A mutant gene; D3999,pSZ5085. The algal transit peptide is underlined, the FLAG epitopetag is uppercase bold and the S111V, V193A residues are lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFvTTPTMRKLRLIWVTARMHIEIYKYPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDaDVRDEYLVHCPRELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHDGDYKDHDIDYKDDDDKSEQ ID NO: 18.-Amino acid sequence of Gm FATA G96A mutant gene;D4000, pSZ5086. The algal transit peptide is underlined, the FLAGepitope tag is uppercase bold and the G96A residue is lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVaCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYKYPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHDGDYKDHDIDYKDDDDKSEQ ID NO: 19-Amino acid sequence of Gm FATA G96T mutant gene;D4001, pSZ5087. The algal transit peptide is underlined, the FLAGepitope tag is uppercase bold and the G96T residue is lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVtCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYKYPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHDGDYKDHDIDYKDDDDKSEQ ID NO: 20-Amino acid sequence of Gm FATA G96V mutant gene;D4002, pSZ5088. The algal transit peptide is underlined, the FLAGepitope tag is uppercase bold and the G96V residue is lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVvCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIETYKYPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHDGDYKDHDIDYKDDDDKSEQ ID NO: 21-Amino acid sequence of Gm FATA G108A mutant gene;D4003, pSZ5089. The algal transit peptide is underlined, the FLAGepitope tag is uppercase bold and the G108A residue is lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTaGFSTTPTMRKLRLIWVTARMHIEIYKYPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHDGDYKDHDIDYKDDDDKSEQ ID NO: 22-Amino acid sequence of Gm FATA L91F mutant gene;D4004, pSZ5090. The algal transit peptide is underlined, the FLAGepitope tag is uppercase bold and the L91F residue is lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANfLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYKYPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHDGDYKDHDIDYKDDDDKSEQ ID NO: 23-Amino acid sequence of Gm FATA L91K mutant gene;D4005, pSZ5091. The algal transit peptide is underlined, the FLAGepitope tag is uppercase bold and the L91K residue is lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANkLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYKYPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHDGDYKDHDIDYKDDDDKSEQ ID NO: 24-Amino acid sequence of Gm FATA L91S mutant gene;D4006, pSZ5092. The algal transit peptide is underlined, the FLAGepitope tag is uppercase bold and the L91S residue is lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANsLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYKYPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHDGDYKDHDIDYKDDDDKSEQ ID NO: 25-Amino acid sequence of Gm FATA G108V mutant gene;D4007, pSZ5093. The algal transit peptide is underlined, the FLAGepitope tag is uppercase bold and the G108V residue is lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTvGFSTTPTMRKLRLIWVTARMHIEIYKYPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHDGDYKDHDIDYKDDDDKSEQ ID NO: 26-Amino acid sequence of Gm FATA T156F mutant gene;D4008, pSZ5094. The algal transit peptide is underlined, the FLAGepitope tag is uppercase bold and the T156F residue is lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYKYPAWSDVVEIESWGQGEGKIGfRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHDGDYKDHDIDYKDDDDKSEQ ID NO: 27-Amino acid sequence of Gm FATA T156A mutant gene;D4009, pSZ5095. The algal transit peptide is underlined, the FLAGepitope tag is uppercase bold and the T156A residue is lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYKYPAWSDVVEIESWGQGEGKIGaRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHDGDYKDHDIDYKDDDDKSEQ ID NO: 28-Amino acid sequence of Gm FATA T156K mutant gene;D4010, pSZ5096. The algal transit peptide is underlined, the FLAGepitope tag is uppercase bold and the T156K residue is lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYKYPAWSDVVEIESWGQGEGKIGkRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHDGDYKDHDIDYKDDDDKSEQ ID NO: 29-Amino acid sequence of Gm FATA T156V mutant gene;D4011, pSZ5097. The algal transit peptide is underlined, the FLAGepitope tag is uppercase bold and the T156V residue is lower-case bold.MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYKYPAWSDVVEIESWGQGEGKIGvRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHDGDYKDHDIDYKDDDDKSEQ ID NO: 30-Nucleotide sequence of the GmFATA wild-type parentalgene expression vector (D3997, pSZ5083). The 5' and 3' homologyarms enabling targeted integration into the Thi4 locus are notedwith lowercase; the CrTUB2 promoter is noted in uppercase italicwhich drives expression of the neomycin selection marker noted withlowercase italic followed by the PmPGH 3'UTR terminator highlightedin uppercase. The PmSAD2-1 promoter (noted in bold text) drivesthe expression of the GmFATA gene (noted with lowercase bold text)and is terminated with the CvNR 3'UTR noted in underlined, lowercase bold. Restriction cloning sites and spacer DNA fragments arenoted as underlined, uppercase plain lettering.ccctcaactgcgacgctgggaaccttctccgggcaggcgatgtgcgtgggtttgcctccttggcacggctctacaccgtcgagtacgccatgaggcggtgatggctgtgtcggttgccacttcgtccagagacggcaagtcgtccatcctctgcgtgtgtggcgcgacgctgcagcagtccctctgcagcagatgagcgtgactttggccatttcacgcactcgagtgtacacaatccatttttcttaaagcaaatgactgctgattgaccagatactgtaacgctgatttcgctccagatcgcacagatagcgaccatgttgctgcgtctgaaaatctggattccgaattcgaccctggcgctccatccatgcaacagatggcgacacttgttacaattcctgtcacccatcggcatggagcaggtccacttagattcccgatcacccacgcacatctcgctaatagtcattcgttcgtgtcttcgatcaatctcaagtgagtgtgcatggatcttggttgacgatgcggtatgggtttgcgccgctggctgcagggtctgcccaaggcaagctaacccagctcctctccccgacaatactctcgcaggcaaagccggtcacttgccttccagattgccaataaactcaattatggcctctgtcatgccatccatgggtctgatgaatggtcacgctcgtgtcctgaccgttccccagcctctggcgtcccctgccccgcccaccagcccacgccgcgcggcagtcgctgccaaggctgtctcggaGGTACC CTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAAC TCTAGAATATCA atgatcgagcaggacggcctccacgccggctcccccgccgcctgggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttctga CAATTGACGCCCGCGCGGCGCACCTGACCTGTTCTCTCGAGGGCGCCTGTTCTGCCTTGCGAAACAAGCCCCTGGAGCATGCGTGCATGATCGTCTCTGGCGCCCCGCCGCGCGGTTTGTCGCCCTCGCGGGCGCCGCGGCCGCGGGGGCGCATTGAAATTGTTGCAAACCCCACCTGACAGATTGAGGGCCCAGGCAGGAAGGCGTTGAGATGGAGGTACAGGAGTCAAGTAACTGAAAGTTTTTATGATAACTAACAACAAAGGGTCGTTTCTGGCCAGCGAATGACAAGAACAAGATTCCACATTTCCGTGTAGAGGCTTGCCATCGAATGTGAGCGGGCGGGCCGCGGACCCGACAAAACCCTTACGACGTGGTAAGAAAAACGTGGCGGGCACTGTCCCTGTAGCCTGAAGACCAGCAGGAGACGATCGGAAGCATCACAGCACAGGATCCCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTCGAAACGTTCACAGCCTAGGGATATC GTGAAAACTCGCTCGACCGCCCGCGTCCCGCAGGCAGCGATGACGTGTGCGTGACCTGGGTGTTTCGTCGAAAGGCCAGCAACCCCAAATCGCAGGCGATCCGGAGATTGGGATCTGATCCGAGCTTGGACCAGATCCCCCACGATGCGGCACGGGAACTGCATCGACTCGGCGCGGAACCCAGCTTTCGTAAATGCCAGATTGGTGTCCGATACCTTGATTTGCCATCAGCGAAACAAGACTTCAGCAGCGAGCGTATTTGGCGGGCGTGCTACCAGGGTTGCATACATTGCCCATTTCTGTCTGGACCGCTTTACCGGCGCAGAGGGTGAGTTGATGGGGTTGGCAGGCATCGAAACGCGCGTGCATGGTGTGTGTGTCTGTTTTCGGCTGCACAATTTCAATAGTCGGATGGGCGACGGTAGAATTGGGTGTTGCGCTCGCGTGCATGCCTCGCCCCGTCGGGTGTCATGACCGGGACTGGAATCCCCCCTCGCGACCCTCCTGCTAACGCTCCCGACTCTCCCGCCCGCGCGCAGGATAGACTCTAGTTCAACCAATCGACA ACT AGTatggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga ATCGATgcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaAAGCTTGAGCTCcagcgccatgccacgccctttgatggcttcaagtacgattacggtgttggattgtgtgtttgttgcgtagtgtgcatggtttagaataatacacttgatttcttgctcacggcaatctcggcttgtccgcaggttcaaccccatttcggagtctcaggtcagccgcgcaatgaccagccgctacttcaaggacttgcacgacaacgccgaggtgagctatgtttaggacttgattggaaattgtcgtcgacgcatattcgcgctccgcgacagcacccaagcaaaatgtcaagtgcgttccgatttgcgtccgcaggtcgatgttgtgatcgtcggcgccggatccgccggtctgtcctgcgcttacgagctgaccaagcaccctgacgtccgggtacgcgagctgagattcgattagacataaattgaagattaaacccgtagaaaaatttgatggtcgcgaaactgtgctcgattgcaagaaattgatcgtcctccactccgcaggtcgccatcatcgagcagggcgttgctcccggcggcggcgcctggctggggggacagctgttctcggccatgtgtgtacgtagaaggatgaatttcagctggttttcgttgcacagctgtttgtgcatgatttgtttcagactattgttgaatgtttttagatttcttaggatgcatgatttgtctgcatgcgactSEQ ID NO: 31-Nucleotide sequence of the GmFATA S111A, V193Amutant gene D3998, pSZ5084). The promoter, 3'UTR, selection markerand targeting arms are the same as described in SEQ ID NO: 30.atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcttcgccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgcggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgaSEQ ID NO: 32-Nucleotide sequence of the GmFATA S111V, V193Amutant gene (D3999, pSZ5085). The promoter, 3'UTR, selection markerand targeting arms are the same as described in SEQ ID NO: 30.atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcttcgtcaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgcggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgaSEQ ID NO: 33-Nucleotide sequence of the GmFATA G96A mutant gene(D4000, pSZ5086). The promoter, 3'UTR, selection marker andtargeting arms are the same as described in SEQ ID NO: 30.atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtggcgtgcaaccacgcccagtccgtgggctactccaccggcggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgaSEQ ID NO: 34-Nucleotide sequence of the GmFATA G96T mutant gene(D4001, pSZ5087). The promoter, 3'UTR, selection marker andtargeting arms are the same as described in SEQ ID NO: 30.atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgacgtgcaaccacgcccagtccgtgggctactccaccggcggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgaSEQ ID NO: 35-Nucleotide sequence of the GmFATA G96V mutant gene(D4002,  pSZ5088). The promoter, 3'UTR, selection marker andtargeting arms are the same as described in SEQ ID NO: 30.atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtggtgtgcaaccacgcccagtccgtgggctactccaccggcggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgaSEQ ID NO: 36-Nucleotide sequence of the GmFATA G108A mutant gene(D4003, pSZ5089). The promoter, 3'UTR, selection marker andtargeting arms are the same as described in SEQ ID NO: 30.atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgccggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgaSEQ ID NO: 37-Nucleotide sequence of the GmFATA L91F mutant gene(D4004, pSZ5090). The promoter, 3'UTR, selection marker andtargeting arms are the same as described in SEQ ID NO: 30.atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacttcctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgaSEQ ID NO: 38-Nucleotide sequence of the GmFATA L91K mutant gene(D4005, pSZ5091).  The promoter, 3'UTR, selection marker andtargeting arms are the same as described in SEQ ID NO: 30.atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacaagctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgaSEQ ID NO: 39-Nucleotide sequence of the GmFATA L91S mutant gene(D4006, pSZ5092). The promoter, 3'UTR, selection marker andtargeting arms are the same as described in SEQ ID NO: 30.atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaactcgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgaSEQ ID NO: 40-Nucleotide sequence of the GmFATA G108V mutant gene(D4007, pSZ5093). The promoter, 3'UTR, selection marker andtargeting arms are the same as described in SEQ ID NO: 30.atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgtcggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgaSEQ ID NO: 41-Nucleotide sequence of the GmFATA T156F mutant gene(D4008, pSZ5094).  The promoter, 3'UTR, selection marker andtargeting arms are the same as described in SEQ ID NO: 30.atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcttccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgaSEQ ID NO: 42-Nucleotide sequence of the GmFATA T156A mutant gene(D4009,  pSZ5095). The promoter, 3'UTR, selection marker andtargeting arms are the same as described in SEQ ID NO: 30.atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcgcgcgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgaSEQ ID NO: 43-Nucleotide sequence of the GmFATA T156K mutant gene(D4010, pSZ5096). The promoter, 3'UTR, selection marker andtargeting arms are the same as described in SEQ ID NO: 30.atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcaagcgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgaSEQ ID NO: 44-Nucleotide sequence of the GmFATA T156V mutant gene(D4011, pSZ5097). The promoter, 3'UTR, selection marker andtargeting arms are the same as described in SEQ ID NO: 30.atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcgtgcgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga SEQ ID NO: 45Amino acid sequence of Gm FATA wild-type parental gene; signalpeptide is removedIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYKYPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRSEQ ID NO: 46Nucleotide sequence of transforming DNA contained in plasmidpSZ5990 transformed into S5780 gctcttcccaactcagataataccaatacccctccttctcctcctcatccattcagtacccccccccttctcttcccaaagcagcaagcgcgtggcttacagaagaacaatcggcttccgccaaagtcgccgagcactgcccgacggcggcgcgcccagcagcccgcttggccacacaggcaacgaatacattcaatagggggcctcgcagaatggaaggagcggtaaagggtacaggagcactgcgcacaaggggcctgtgcaggagtgactgactgggcgggcagacggcgcaccgcgggcgcaggcaagcagggaagattgaagcggcagggaggaggatgctgattgaggggggcatcgcagtctctcttggacccgggataaggaagcaaatattcggccggttgggttgtgtgtgtgcacgttttcttcttcagagtcgtgggtgtgcttccagggaggatat

ttcctgttcctgctggccggcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaagTGA caattgacgcccgcgcggcgcacctgacctgttctctcgagggcgcctgttctgccttgcgaaacaagcccctggagcatgcgtgcatgatcgtctctggcgccccgccgcgcggtttgtcgccctcgcgggcgccgcggccgcgggggcgcattgaaattgttgcaaaccccacctgacagattgagggcccaggcaggaaggcgttgagatggaggtacaggagtcaagtaactgaaagtttttatgataactaacaacaaagggtcgtttctggccagcgaatgacaagaacaagattccacatttccgtgtagaggcttgccatcgaatgtgagcgggcgggccgcggacccgacaaaacccttacgacgtggtaagaaaaacgtggcgggcactgtccctgtagcctgaagaccagcaggagacgatcggaagcatcacagcaca ggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatc

catcgccgccgccgccgtgatcgtgcccctgggcctgctgttcttcatctccggcctggtggtgaacctgatccaggccctgtgcttcgtgctgatccgccccctgtccaagaacacctaccgcaagatcaaccgcgtggtggccgagctgctgtggctggagctgatctggctggtggactggtgggccggcgtgaagatcaaggtgttcatggaccccgagtccttcaacctgatgggcaaggagcacgccctggtggtggccaaccaccgctccgacatcgactggctggtgggctggctgctggcccagcgctccggctgcctgggctccgccctggccgtgatgaagaagtcctccaagttcctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgctcctgggccaaggacgagaacaccctgaaggccggcctgcagcgcctgaaggacttcccccgccccttctggctggccttcttcgtggagggcacccgcttcacccaggccaagttcctggccgcccaggagtacgccgcctcccagggcctgcccatcccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtcccacatgcgctccttcgtgcccgccatctacgacatgaccgtggccatccccaagtcctccccctcccccaccatgctgcgcctgttcaagggccagccctccgtggtgcacgtgcacatcaagcgctgcctgatgaaggagctgcccgagaccgacgaggccgtggcccagtggtgcaaggacatgttcgtggagaaggacaagctgctggacaagcacatcgccgaggacaccttctccgaccagcccatgcaggacctgggccgccccatcaagtccctgctggtggtggcctcctgggcctgcctgatggcctacggcgccctgaagttcctgcagtgctcctccctgctgtcctcctggaagggcatcgccttcttcctggtgggcctggccatcgtgaccatcctgatgcacatcctgatcctgttctcccagtccgagcgctccacccccgccaaggtggcccccggcaagcccaagaacgacggcgagacctccgaggcccgccgcgacaagcagcagTGA atgcatatgtggagatgtagggtggtcgactcgttggaggtgggtgtttttttttatcgagtgcgcggcgcggcaaacgggtccctttttatcgaggtgttcccaacgccgcaccgccctcttaaaacaacccccaccaccacttgtcgaccttctcgtttgttatccgccacggcgccccggaggggcgtcgtctggccgcgcgggcagctgtatcgccgcgctcgctccaatggtgtgtaatcttggaaagataataatcgatggatgaggaggagagcgtgggagatcagagcaaggaatatacagttggcacgaagcagcagcgtactaagctgtagcgtgttaagaaagaaaaactcgctgttaggctgtattaatcaaggagcgtatcaataattaccgaccctatacctttatctccaacccaatcgcggcttaag gatctaagtaagattcgaagcgctcgaccgtgccggacggactgcagccccatgtcgtagtgaccgccaatgtaagtgggctggcgtttccctgtacgtgagtcaacgtcactgcacgcgcaccaccctctcgaccggcaggaccaggcatcgcgagatacagcgcgagccagacacggagtgccgagctatgcgcacgctccaact

tgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcg

atccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtggcgtgcaaccacgcccagtccgtgggctactccaccgccggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgc

cgatggagcgacgagtgtgcgtgcggggctggcgggagtgggacgccctcctcgctcctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagatccggccgcacatcaaagggcccctccgccagagaagaagctcctttcccagcagactccttctgctgccaaaacacttctctgtccacagcaacaccaaaggatgaacagatcaacttgcgtctccgcgtagcttcctcggctagcgtgcttgcaacaggtccctgcactattatcttcctgctttcctctgaattatgcggcaggcgagcgctcgctctggcgagcgctccttcgcgccgccctcgctgatcgagtgtacagtcaatgaatggtgagctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaaccgaatgctgcgtgaacgggaaggaggaggagaaagagtgagcagggagggattcagaaatgagaaatgagaggtgaaggaacgcatccctatgcccttgcaatggacagtgtttctggccaccgccaccaagacttcgtgtcctctgatcatcatgcgattgattacgttgaatgcgacggccggtcagccccggacctccacgcaccggtgctcctccaggaagatgcgcttgtcctccgccatcttgcagggctcaagctgctcccaaaactcttgggcgggttccggacggacggctaccgcgggtgcggccctgaccgccactgttcggaagcagcggcgctgcatgggcagcggccgctgcggtgcgccacggaccgcatgatccaccggaaaagcgcacgcgctggagcgcgcagaggaccacagagaagcggaagagacgccagtactggcaagcaggctggtcggtgccatggcgcgctactaccctcgctatgactcgggtcctcggccggctggcggtgctgacaattcgtttagtggagcagcgactccattcagctaccagtcgaactcagtggcacagtgactccc gctctt cSEQ ID NO: 47-Nucleotide sequence of CpSAD transit peptide fusedto GarmFATA1 (G108A) gene and 3X FLAG tag in pSZ5936 and pSZ6018 actagtATGgccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgcc atccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgccggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgc atggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaag TGA atcgatSEQ ID NO: 48-Nucleotide sequence of CpSAD transit peptide fusedto GarmFATA1 (G96A, S111A) gene and 3X FLAG tag in pSZ5991 and pSZ6026actagt ATGgccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgcc atccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtggcgtgcaaccacgcccagtccgtgggctactccaccggcggcttcgccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgc atggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaag TGA atcgatSEQ ID NO: 49-Nucleotide sequence of CpSAD transit peptide fusedto GarmFATA1 (G96A, V193A) gene and 3X FLAG tag in pSZ5986 and pSZ6023actagt ATGgccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgcc atccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtggcgtgcaaccacgcccagtccgtgggctactccaccggcggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgcggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgc atggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaag TGA atcgatSEQ ID NO: 50-Nucleotide sequence of CpSAD transit peptide fusedto GarmFATA1 (G108A, S111A) gene and 3X FLAG tag in pSZ5982 and pSZ6019actagt ATGgccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgcc atccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgccggcttcgccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgc atggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaag TGA atcgatSEQ ID NO: 51-Nucleotide sequence of CpSAD transit peptide fusedto GarmFATA1 (G108A, V193A) gene and 3X FLAG tag in pSZ5983 and pSZ6020actagt ATGgccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgcc atccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgccggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgcggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgc atggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaag TGA atcgatSEQ ID NO: 52-Nucleotide sequence of CpSAD transit peptide fusedto GarmFATA1 (G96A, G108A, S111A) gene and 3X FLAG tag in pSZ6005and pSZ6028 actagtATGgccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgcc atccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtggcgtgcaaccacgcccagtccgtgggctactccaccgccggcttcgccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgc atggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaag TGA atcgatSEQ ID NO: 53-Nucleotide sequence of CpSAD transit peptide fusedto GarmFATA1 (G96A, G108A, V193A) gene and 3X FLAG tag in pSZ5984and pSZ6021 actagtATGgccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgcc atccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtggcgtgcaaccacgcccagtccgtgggctactccaccgccggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgcggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgc atggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaag TGA atcgatSEQ ID NO: 54-Nucleotide sequence of CpSAD transit peptide fusedGarmFATA1 (G96A, S111A, V193A) gene and 3X FLAG tag in pSZ6004 actagtATGgccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgcc atccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtggcgtgcaaccacgcccagtccgtgggctactccaccggcggcttcgccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgcggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgc atggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaag TGA atcgatSEQ ID NO: 55-Nucleotide sequence of CpSAD transit peptide fusedto GarmFATA1 (G108A, S111A, V193A) gene and 3X FLAG tag in pSZ5985and pSZ6022 actagtATGgccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgc gggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgccggcttcgccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgcggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgc atggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaag TGA atcgatSEQ ID NO: 56 Nucleotide sequence of CpSAD transit peptide fusedto GarmFATA1 (G96A, G108A, S111A, V193A) gene and 3X FLAG tag in pSZ5987actagt ATGgccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgcc atccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtggcgtgcaaccacgcccagtccgtgggctactccaccgccggcttcgccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgcggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgc atggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaag TGA atcgatSEQ ID NO: 57-Nucleotide sequence of PmACP-P1 promoter in pSZ6019-pSZ6023, pSZ6026 and pSZ6028

What is claimed is:
 1. A non-natural fatty acyl-ACP thioesterase,wherein the non-natural thioesterase has at least 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 45 andcomprises an alanine (A) at the position corresponding to position 74 ofSEQ ID NO: 45, wherein the non-natural thioesterase catalyzes theproduction of increased levels of stearate (C18:0) in comparison to awild-type thioesterase.
 2. The non-natural thioesterase of claim 1,further wherein the non-natural thioesterase comprises an alanine (A) atthe position corresponding to position 89 of SEQ ID NO: 45 and/or analanine (A) at the position corresponding to position 171 of SEQ ID NO:45, and/or an alanine (A) at the position corresponding to position 86of SEQ ID NO:
 45. 3. A method for producing a triglyceride oil, themethod comprising expressing, in a host cell, the non-naturalthioesterase of claim 1; cultivating the host cell; and isolating theoil.
 4. A method for increasing the C18:0 fatty acids in a fatty acidprofile of an oil produced by an optionally oleaginous host cell, themethod comprising, providing a parent gene encoding a fatty acyl-ACPthioesterase B (FATB) enzyme, mutating the gene to so as to encode anon-natural thioesterase having at least 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 45 and comprising analanine (A) at the position corresponding to position 74 of SEQ ID NO:45; expressing the mutated gene in the host cell; and producing the oil,whereby the C18:0 fatty acids in the fatty acid profile of the oil areincreased.
 5. The method of claim 4, further mutating the gene to so asto encode a non-natural thioesterase having an alanine (A) at theposition corresponding to position 89 of SEQ ID NO: 45 and/or an alanine(A) at the position corresponding to position 171 of SEQ ID NO: 45,and/or an alanine (A) at the position corresponding to position 86 ofSEQ ID NO:
 45. 6. A method for producing a triglyceride oil, the methodcomprising expressing, in a host cell, the non-natural thioesterase ofclaim
 2. 7. An isolated polynucleotide encoding a non-natural fattyacyl-ACP thioesterase having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identity to SEQ ID NO: 45 and comprises an alanine(A) at the position corresponding to position 74 of SEQ ID NO: 45,wherein the non-natural thioesterase catalyzes the production ofincreased levels of stearate (C18:0) in comparison to a wild-typethioesterase.
 8. The isolated polynucleotide of claim 7, encoding anon-natural thioesterase further comprising an alanine (A) at theposition corresponding to position 89 of SEQ ID NO: 45 and/or an alanine(A) at the position corresponding to position 171 of SEQ ID NO: 45,and/or an alanine (A) at the position corresponding to position 86 ofSEQ ID NO:
 45. 9. An expression cassette comprising the isolatedpolynucleotide of claim
 7. 10. A host cell comprising the expressioncassette of claim
 9. 11. The host cell of claim 10, wherein the cell isa oleaginous microalga cell.
 12. The host cell of claim 11, wherein thecell is a Prototheca cell.
 13. The host cell of claim 12, wherein thecell is a Prototheca moriformis cell.
 14. A method for producing atriglyceride oil, the method comprising expressing, in a host cell, theisolated polynucleotide of claim 7; cultivating the host cell; andisolating the oil.
 15. An expression cassette comprising the isolatedpolynucleotide of claim
 8. 16. A host cell comprising the expressioncassette of claim
 15. 17. The host cell of claim 16, wherein the cell isa oleaginous microalga cell.
 18. The host cell of claim 17, wherein thecell is a Prototheca cell.
 19. The host cell of claim 18, wherein thecell is a Prototheca moriformis cell.
 20. A method for producing atriglyceride oil, the method comprising expressing, in a host cell, theisolated polynucleotide of claim 8; cultivating the host cell; andisolating the oil.